Composition for obtaining protein-rich nutrient supplements from bacterial fermentation process

ABSTRACT

Protein-rich nutrient supplements and animal feed supplements derived from an anaerobic bacterial process are generated through a myriad of cell rupturing and protein fractionation/purification processes. Bacterial fermentation systems and methods of obtaining one or more protein-containing portions from a fermentation process using carbon monoxide-containing gaseous substrates are provided. The invention further provides compositions of protein-rich nutrient supplements with useful applications for intake by a variety of different animals and humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent applicationSer. No. 62/674,604, filed May 21, 2018, the above-referencedapplication is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Microbial fermentation occurs when a microorganism is provided with acarbon substrate that it can utilize and process into various products,which can be recovered, separated, and purified. Chosen carbonsubstrates depend on the type of microorganisms used and their metabolicpathways, and the type of microorganisms used are based on identifyingand selecting a microbial strain that has the capabilities to bring thetype of desired products. Carbon substrates can include carbon monoxide(CO), carbon dioxide (CO₂), methanol, methyl, ethanol, n-alkanes,glucose, cellulose, bagasse, molasses, and sulfite waste. Usefulproducts and substances generated by bacterial fermentation includeethanol, lactic acid, acetate, and other biofuels and chemicals, whichcan be used as a source of energy and a variety of additionalapplications.

As an example, bacterial fermentation by anaerobic microorganisms,including acetogenic microorganisms, may produce fermentation products(e.g., ethanol, butanol, acetate, butyrate, butyric acid,2,3-butanediol, and other related products) through fermentation ofgaseous substrates such as carbon monoxide (CO), hydrogen gas (H₂),and/or carbon dioxide (CO₂). Ethanol and butanol are often used asliquid fuels relating to transportation, whereas acetate and2,3-butanediol are used in the chemical industry. Examples ofbioethanol-producing acetogens used for microbial fermentation includethose from the genus Clostridium and Acetobacterium. For example, U.S.Pat. No. 5,173,429 describes Clostridium ljungdahlii ATCC No. 49587, ananaerobic microorganism that produces ethanol and acetate from synthesisgas. U.S. Pat. No. 5,807,722 describes a method and apparatus forconverting waste gases into organic acids and alcohols using Clostridiumljungdahlii ATCC No. 55380. U.S. Pat. No. 6,136,577 describes a methodand apparatus for converting waste gases into ethanol using Clostridiumljungdahlii ATCC No. 55988 and 55989.

In addition to the fermentation products, large scale microbialfermentation also produces a large amount of microbial fermentationculture broth and may require purging of a large portion of the dead orinactive cells. Recovery of excess bacterial cells from excess or purgedcultural broth into microbial biomass can lead to the generation ofsingle cell proteins (SCP) and other components for re-use as source ofproteins, amino acids, and carbohydrates that are useful as a feedstockfor an animal feed, and/or animal feed nutrients or supplements. Allanimals require amino acids, the building blocks of proteins necessaryfor optimal growth, reproduction, lactation, and maintenance. Aminoacids absorbed in the cow's small intestine are derived from proteinsthat are digested in the rumen and generally its digestion system mustsupply 10 essential amino acids, which cannot be self-produced by thecow, including arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan, and valine. Ideally,the relative proportions of each of the essential amino acids absorbedwould exactly match the cow's required amino acid supply, because ashortage of one can limit the utilization of others.

However, current methods targeting single cell proteins often directlyincorporate microbial cells as whole cell biomass to be used for animalfeed or aquaculture. In microbial fermentation processes, thefermentation broth includes bacterial cells as well as cell debris.These methods do not differentiate the two, and often contain biomasscontents which may be harmful to the animal or aquaculture (e.g., fishesor shrimps, etc). For example, microbial whole cell biomass may containhigh nucleic acid content that is not suitable for ingestion or othercontents that cannot not be properly digested. Most of these priormethods do not process the whole cell biomass by additionalcell-rupturing or cell disruption techniques prior to incorporation thewhole cell biomass into animal feeds. In addition, current methods ofrecovering bacterial proteins from bacterial fermentation do not yieldhigh enough protein content suitable for nutrition-related purposes.There is a need for a method and a system for obtaining protein-richsupplements from a bacterial fermentation process, and composition ofany such nutrient supplements and animal feeds.

SUMMARY OF THE INVENTION

Embodiments of the invention provide methods, systems, and compositionsfor producing and obtaining protein-rich nutrient supplements and/oranimal feeds that are derived from microbial cell biomass after ananaerobic bacterial fermentation process using a myriad of cellrupturing and protein fractionation and purification techniques. Theprotein-rich nutrient supplements can be used as feedstock directly ortogether with other nutrients as supplements for human or animals.

In one embodiment, a bacterial fermentation system producing aprotein-containing portion from a fermentation process is provided andincludes one or more fermentation vessels, one or more cell separators,one or more processing chambers, one or more cell rupturing devices, andone or more fractionators. In another embodiment, the invention furtherprovides a composition of a protein-rich nutrient supplement generatedfrom a fermentation process using anaerobic bacteria with usefulapplications for intake by a variety of different animals and humans.

In still another embodiment, a method is provided for extracting aprotein-rich portion out of microbial cell biomass from an anaerobicbacterial fermentation and using the protein-rich portion as a nutrientsupplement. In one aspect, the method includes fermenting a solid,liquid or gaseous substrate with an anaerobic bacteria in a fermentationvessel containing a liquid culture medium, obtaining from thefermentation vessel an amount of a fermentation liquid broth containinga first batch of anaerobic bacterial cells at a first concentration,separating the cells of the anaerobic bacteria from the fermentationliquid broth into a cell-free permeate solution and a cell-containingsuspension containing a second batch of anaerobic bacterial cells at asecond concentration. In one example, the second concentration of theanaerobic bacterial cells in the second batch is higher than the firstconcentration of the anaerobic bacterial cells in the first batch. Oncethe cell-containing suspension is obtained, the method further includesrupturing cell membranes of the anaerobic bacterial cells within thecell-containing suspension to generate a homogenate, fractionating thehomogenate into a first protein-containing portion and aprotein-containing cell debris portion using one or more fractionators;and obtaining the first protein-containing portion.

In one aspect, the first protein-containing portion and/or theprotein-containing cell debris portion can be processed and produced asprotein-rich nutrient supplements. In another aspect, the firstprotein-containing portion is produced as a protein-rich nutrientsupplement having a protein content of about 10% or larger, such as 40%or larger, 50% or larger, 60% or larger, 70% or larger, or 80% orgreater, 90% or greater, such as between about 10% to about 80% ofprotein content, or between about 10% to about 95% of protein content,e.g., between about 10% to about 98% of protein content.

In still another aspect, the method may also include holding thecell-containing suspension containing the anaerobic bacterial cells in acell-containing holding tank and delivering the cell-containingsuspension from the cell-containing holding tank at a delivery rate to arupturing device. The cell-containing holding tank can serve as astorage vessel or a pretreatment chamber for the cell-containingsuspension. In one example, the cell-containing holding tank is used toconduct a pretreatment step of treating the cell-containing suspensioncontaining the anaerobic bacterial cells with one or more additivessupplied through an inlet line that is connected to the cell-containingholding tank. Examples of the additives include, but are not limited toa surfactant, detergent, EDTA, Tween-20, Triton X-100, sodium dodecylsulfate, CHAPS, an enzyme, protease, lysozyme, benzonase, nuclease, apH-adjusting agent, and a combination thereof.

In another aspect, the method further includes treating thecell-containing suspension containing the anaerobic bacterial cells withone or more additives prior to the rupturing of the cell membranes ofthe anaerobic bacterial cells. Alternatively, the method includestreating the cell-containing suspension containing the anaerobicbacterial cells with one or more additives prior to the separating thefirst protein-containing portion from the cell-containing cell debrisportion.

In still another aspect, the method may further include concentratingthe cell-containing suspension containing the cells of the anaerobicbacteria into a second cell-containing suspension. In one example, thesecond cell-containing suspension is delivered to a cell-containingholding tank to be concentrated and/or stored therein. In anotherexample, the second cell-containing suspension in the holding tank issubjected to a pretreatment step of treating the second cell-containingsuspension containing the anaerobic bacterial cells with one or moreadditives supplied through an inlet connected to the cell-containingholding tank. Then, the second cell-containing suspension is deliveredout of the holding tank into a rupturing device. The rupturing deviceruptures the cell membranes of the anaerobic bacterial cells within thesecond cell-containing suspension and generates a homogenate. Additionalprotein-containing portions are then separated from a cell-containingcell debris portion within the homogenate.

In still another aspect, the method may further include delivering thefirst protein-containing portion to one or more fractionators,fractionating the first protein-containing portion into a secondprotein-containing portion and/or a third or more protein-containingportion using the one or more fractionators, and collecting the secondand the third or more protein-containing portions. Example offractionators includes, but are not limited to, a solid-liquidfractionator, a centrifugation device, a continuous centrifuge, adecanter centrifuge, a disc-stack centrifuge, a filtration device, ahollow fiber filtration device, a spiral wound filtration device, aceramic filter device, a cross-flow filtration device, a size exclusiondevice, one or series of size exclusion columns, one or series ionexchange columns, one or series of carbon polymer columns, aflow-through magnetic fractionator, an ultrafiltration device, one orseries of affinity chromatography columns, one or series of gelfiltration columns, and combinations thereof.

In one embodiment, the first protein-containing portion can be deliveredto a filtration device to be filtered through the filtration device andfractionated into a retentate portion and a filtrate portion so that thefiltrate portion is produced as the protein-rich nutrient supplement. Inanother embodiment, the retentate portion is produced as theprotein-rich nutrient supplement. In still another embodiment, the firstprotein-containing portion can be delivered to a centrifuge andfractionated into a supernatant protein-containing portion and a pelletprotein-containing portion by centrifugation so that the supernatantprotein-containing portion and/or the pellet protein-containing portionare produced as the protein-rich nutrient supplement.

Another embodiment of the invention provides a bacterial fermentationsystem for producing a protein-rich nutrient supplement from ananaerobic bacterial fermentation process. The bacterial fermentationsystem includes a fermentation vessel connected to a gas inlet line forflowing a gaseous substrate into the fermentation vessel and a liquidinlet line for supplying a culture medium into the fermentation vesselcontaining the anaerobic bacteria to ferment the gaseous substrate andthe culture medium into a fermentation liquid broth, one or more firstcell separators connected to a first outlet line of the fermentationvessel to receive a first flow of the fermentation liquid broth from thefermentation vessel and separate the first flow of the fermentationliquid broth into a first cell-containing suspension and a firstcell-free permeate solution, and a processing chamber connected to asecond outlet line of the first cell separator to receive the firstcell-free permeate solution from the first cell separator and processthe first cell-free permeate solution into an oxygenatedhydrocarbonaceous compound.

The bacterial fermentation system further includes one or more cellrupturing devices to receive the first cell-containing suspension,rupture cell membranes of cells contained within the firstcell-containing suspension, and generate a homogenate, and one or morefractionators connected to an outlet line of the one or more cellrupturing devices to receive the homogenate from the one or more cellrupturing devices and fractionate the homogenate into a firstprotein-containing portion and a protein-containing cell debris portion.

In one embodiment, cell rupturing is accomplished by a microfluidizer.In another embodiment, cell rupturing is accomplished using a sonicator.In still another embodiment, cell rupturing is accomplished using amicrofluidizer with a processing pressure in the range of 5,000 to25,000 pounds per square inch (psi). In still another embodiment, cellrupturing is accomplished using a microfluidizer with a processingpressure in the range of 15,000 to 20,000 pounds per square inch (psi).In yet another embodiment, cell rupturing is accomplished using amicrofluidizer with a processing pressure of 15,000 pounds per squareinch (psi).

In one aspect, the bacterial fermentation system includes a first cellseparator connected to the fermentation vessel and a rupturing device.The first cell separator receives a fermentation liquid at a first cellconcentration from the fermentation vessel and separates thefermentation liquid into a cell-free permeate solution and acell-containing suspension at a second cell concentration. In anotheraspect, the bacterial fermentation system further includes acell-containing holding tank connected to the first cell separator toreceive an amount of the first cell-containing suspension. In stillanother aspect, the bacterial fermentation system further includes asecond cell separator connected to a fourth outlet line of thefermentation vessel to receive a second flow of the fermentation liquidbroth from the fermentation vessel and separate the second flow of thefermentation liquid broth into a second cell-containing suspension and asecond cell-free permeate solution.

After one or more cell-containing suspensions are processed and rupturedinto the homogenate, the homogenate is fractionated by the one or morefractionators into one or more protein-containing portions and one ormore protein-containing cell debris portions. In addition, the one ormore cell-containing suspensions can be delivered from one or moreoutlet of one or more cell separators to the rupturing device and/or acell-containing holding tank, wherein the cell-containing holding tankcan either hold, house or concentrate the one or more cell-containingsuspensions and delivers the one or more cell-containing suspensions ata delivery rate to the rupturing device or one or more fractionators forfurther processing.

In another aspect, after fermentation of the gaseous substrate by theanaerobic bacteria, the first cell separator receives a first flow of afirst fermentation liquid broth containing bacterial cells. A secondcell separator connected to the fermentation vessel receives a secondflow of a second fermentation liquid broth containing bacterial cells.The second cell separator separates the second fermentation liquid brothinto a second cell-free permeate solution and a second cell-containingsuspension containing anaerobic bacterial cells. In this aspect, arupturing device is used to receive the second cell-containingsuspension from the second cell separator, rupture the cell membranes ofthe second cell-containing suspension and generate a homogenate. Inanother aspect, a holding tank receives the second cell-containingsuspension from the second cell separator and sends the secondcell-containing suspension to the rupturing device.

In yet another aspect, the bacterial fermentation system provides one ormore rupturing devices to rupture the cell membranes of anaerobicbacterial cells within the cell-containing suspension. Examples of therupturing devices include, but are not limited to, a microfluidicsdevice, a sonication device, an ultrasonic device, a mechanicaldisruption device, a French press, a freezer, a heater, a heatexchanger, a distillation column, a device that increases thetemperature of process streams and holding tanks, a pasteurizationdevice, an UV sterilization device, a gamma ray sterilization device, areactor, a homogenizer, and combinations thereof.

In another embodiment, the present invention is a composition of aprotein-rich nutrient supplement generated from a fermentation processusing an acetogenic bacterial culture. In one aspect, the compositionincludes a protein-containing portion fractionated from a homogenate,wherein the homogenate is obtained from rupturing a cell-containingsuspension containing cells of the anaerobic bacteria, and thecell-containing suspension is obtained from a fermentation liquid brothbeing delivered out of a fermentation vessel during fermentation of agaseous substrate by the anaerobic bacteria, wherein cells of theanaerobic bacteria from the fermentation liquid broth are separated intothe cell-containing suspension and a cell-free permeate solution. Inanother aspect, the composition includes a protein-containing celldebris portion fractionated from a homogenate.

In still another aspect, the composition includes a fermentation-derivedprotein, wherein the fermentation-derived protein is from fermentationof a solid, liquid or gaseous substrate in a liquid culture medium withnatural-occurring or genetically modified acetogenic bacteria of thegenus, Clostridium, Acetobacterium, Butyribacterium, Eubacterium, andsimilar variants thereof. The substrate comprises of one or more liquid,solid or gases that include, but are not limited to, carbohydrates,carboxylic acids, methanol, methane, carbon monoxide, (CO), carbondioxide (CO₂), hydrogen (H₂) gas, nitrogen gas (N₂), syngas, andcombinations thereof. In one embodiment, the fermentation isaccomplished using a naturally occurring or non-naturally occurringmethanotrophic bacteria.

As yet another embodiment, the composition includes a purified proteinproduct fractionated from a first amount of a protein-containing portionof a homogenate, and a second amount of a protein-containing cell debrisportion of a homogenate, wherein the homogenate is obtained fromrupturing a cell-containing suspension containing cells of theacetogenic bacterial culture, and wherein the cell-containing suspensionis obtained from a fermentation liquid being delivered out of afermentation vessel during fermentation of a gaseous substrate using theacetogenic bacterial culture.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the presentinvention can be understood in detail, a more particular description ofthe invention, briefly summarized above, may be had by reference toembodiments, some of which are illustrated in the appended drawings. Itis to be noted, however, that the appended drawings illustrate onlytypical embodiments of this invention and are therefore not to beconsidered limiting of its scope, for the invention may admit to otherequally effective embodiments.

FIG. 1A illustrates a flow chart of a method of processing acell-containing suspension from a fermentation process having a cultureof an anaerobic bacteria therein and obtaining a firstprotein-containing portion and/or a protein-containing cell debrisportion as a protein-rich nutrient supplement according to one or moreembodiments of the invention.

FIG. 1B illustrates a flow chart of another method of processing acell-containing suspension from a fermentation process having a cultureof an anaerobic bacteria therein and obtaining a firstprotein-containing portion and/or a protein-containing cell debrisportion as a protein-rich nutrient supplement according to one or moreembodiments of the invention.

FIG. 2A illustrates a flow chart of a method of processing acell-containing suspension from a fermentation process having a cultureof an anaerobic bacteria therein and obtaining a secondprotein-containing portion as a protein-rich nutrient supplementaccording to one or more embodiments of the invention.

FIG. 2B illustrates a flow chart of another method of processing acell-containing suspension from a fermentation process having a cultureof an anaerobic bacteria therein and obtaining a thirdprotein-containing portion as a protein-rich nutrient supplementaccording to one or more embodiments of the invention.

FIG. 3A illustrates a schematic of a bacterial fermentation system 300Afor producing a cell-containing suspension and one or more oxygenatedhydrocarbonaceous compounds from a fermentation process using a cultureof an anaerobic bacteria, where the bacterial fermentation system 300Aincludes one or more cell separators, one or more processing chambers,and optionally, one or more dehydration chambers, according to one ormore embodiments of the invention.

FIG. 3B illustrates a schematic of a bacterial fermentation system 300Bfor producing one or more cell-containing suspensions and one or moreoxygenated hydrocarbonaceous compounds from a fermentation process usinga culture of an anaerobic bacteria, where the bacterial fermentationsystem 300B includes two cell separators, a cell-free holding tank, aprocessing chamber, and optionally, a dehydration chamber, according toone or more embodiments of the invention.

FIG. 4A shows a schematic of a bacterial fermentation system 400A withone or more cell separators, one or more processing chambers, one ormore rupturing devices, one or more fractionators, and one or moredehydration chambers for a fermentation process using a culture of ananaerobic bacteria according to one or more embodiments of theinvention.

FIG. 4B shows a schematic of a bacterial fermentation system 400B withtwo cell separators, one processing chamber, one rupturing device, twofractionators and three dehydration chambers for a fermentation processusing a culture of an anaerobic bacteria according to one or moreembodiments of the invention.

FIG. 4C shows a schematic of a bacterial fermentation system 400C withone cell separator, one cell-free holding tank, one processing chamber,one rupturing device, one fractionator, and two or more dehydrationchambers for a fermentation process using a culture of an anaerobicbacteria according to one or more embodiments of the invention.

FIG. 4D shows a schematic of a bacterial fermentation system 400D withone cell separator, one cell-free holding tank, one processing chamber,one rupturing device, two fractionators, and optionally, additionaldehydration chambers for a fermentation process using a culture of ananaerobic bacteria according to one or more embodiments of theinvention.

FIG. 4E shows a schematic of a bacterial fermentation system 400E withtwo cell separators, one cell-free holding tank, one processing chamber,one rupturing device, two fractionators, and optionally, threedehydration chambers for a fermentation process using a culture of ananaerobic bacteria according to one or more embodiments of theinvention.

FIG. 4F shows a schematic of a bacterial fermentation system 400F withone cell separator, one cell-free holding tank, one processing chamber,one cell-containing holding tank, one rupturing device, twofractionators, and optionally, three dehydration chambers for afermentation process using a culture of an anaerobic bacteria accordingto one or more embodiments of the invention.

FIG. 4G shows a schematic of a bacterial fermentation system 400G withtwo cell separators, one cell-free holding tank, one processing chamber,one cell-containing holding tank, one rupturing device, twofractionators, and optionally, three dehydration chambers for afermentation process using a culture of an anaerobic bacteria accordingto one or more embodiments of the invention.

FIG. 4H shows a schematic of a bacterial fermentation system 400H withtwo cell separators, one cell-free holding tank, one processing chamber,one rupturing device, two fractionators, and optionally, threedehydration chambers for a fermentation process using a culture of ananaerobic bacteria according to one or more embodiments of theinvention.

FIG. 5A is a schematic of an exemplary bacterial fermentation system forrupturing cells collected from an anaerobic bacterial fermentationprocess and obtaining one or more protein-containing portions fromhomogenates according to one or more embodiments of the invention.

FIG. 5B is a schematic of another exemplary bacterial fermentationsystem for rupturing cells collected from an anaerobic bacterialfermentation process and obtaining one or more protein-containingportions from homogenates according to one or more embodiments of theinvention.

FIG. 5C is a schematic of another example of a bacterial fermentationsystem for rupturing cells collected from an anaerobic bacterialfermentation process and obtaining one or more protein-containingportions from homogenates according to one or more embodiments of theinvention.

FIG. 5D is a schematic of yet another example of a bacterialfermentation system for rupturing cells collected from an anaerobicbacterial fermentation process and obtaining one or moreprotein-containing portions from homogenates according to one or moreembodiments of the invention.

FIG. 5E is a schematic of another exemplary bacterial fermentationsystem for rupturing cells collected from an anaerobic bacterialfermentation process and obtaining one or more protein-containingportions from homogenates according to one or more embodiments of theinvention.

FIG. 6 is a schematic of still another example of a bacterialfermentation system for rupturing cells collected from an anaerobicbacterial fermentation process and obtaining one or moreprotein-containing portions from homogenates according to one or moreembodiments of the invention.

FIG. 7A shows an electronic micrograph of one example of acell-containing suspension before rupturing anaerobic bacterial cellswithin the cell-containing suspension into a homogenate, according toone or more embodiments of the invention.

FIG. 7B shows an electronic micrograph of one example of a homogenateobtained from a rupturing device after rupturing anaerobic bacterialcells within a cell-containing suspension of a bacterial fermentationliquid broth, according to one or more embodiments of the invention.

FIG. 7C is still another example of an electronic micrograph, showingthe anaerobic bacterial cells within the cell-containing suspensionafter it is ruptured inside the rupturing devices, according to one ormore embodiments of the invention.

FIG. 7D is another example of an electronic micrograph of a homogenate,showing ruptured cell membranes of the anaerobic bacterial cells withinthe cell-containing suspension after ruptured by the rupturing device athigh pressure, according to one or more embodiments of the invention.

FIG. 7E is still another example of an electronic micrograph of ahomogenate, showing ruptured cell membranes of the anaerobic bacterialcells within the cell-containing suspension after ruptured by therupturing device at very high pressure, according to one or moreembodiments of the invention, according to one or more embodiments ofthe invention.

FIG. 8A shows a graph of an example of soluble protein concentrations inhomogenate obtained from a rupturing device after rupturing anaerobicbacterial cells within an example of a cell-containing suspension,according to one or more embodiments of the invention.

FIG. 8B shows another graph of another example of soluble proteinconcentrations obtained from a rupturing device after rupturing cellmembranes of the anaerobic bacterial cells within another example of acell-containing suspension, according to one or more embodiments of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention provide methods, systems, and compositionsfor producing and obtaining protein-rich nutrient supplements and/oranimal feeds that are derived from microbial cell biomass after ananaerobic bacterial fermentation process using a myriad of cellrupturing and protein fractionation and purification techniques. Morespecifically, the invention relates to a method of separating amicrobial biomass out of a fermentation process, rupturing the cells ofthe microbial biomass into a homogenate and fractionating and purifyingone or more protein-containing portions from the homogenate so that theone or more protein-containing portions can be further processed into acomposition as a nutrient supplement ingestible by both animals andhumans, The protein-rich nutrient supplements can be used as feedstockdirectly or together with other nutrients as supplements for human oranima

Protein-rich nutrient supplements and animal feed supplements can beprocessed and obtained from one or more protein-containing portionsafter a fermentation process in a bacterial fermentation system usingone or more gaseous substrates, such as syngas, carbon sourcesubstrates, carbon monoxide (CO)-containing gas, carbon dioxide (CO₂),hydrogen gas (H₂), syngas, and combinations thereof. The inventionfurther provides compositions of protein-rich nutrient supplements withuseful applications for intake by animals and humans.

I. Processing of Microbial Biomass to Generate Fermentation-DerivedProteins

A bacterial fermentation process generally includes fermenting a gaseoussubstrate, such as syngas or carbon monoxide (CO)-containing gaseoussubstrate by a bacteria, such as an anaerobic bacteria or an acetogenicbacteria, among others, and generating fermentation products whichinclude carbon dioxide (CO2), ethanol, butanol, butyric acid, aceticacid, etc. More importantly, after an anaerobic bacterial fermentationprocess, large amounts of microbial biomass are obtained. The largeamounts of microbial biomass can be purged during or after the bacterialfermentation process. Upon completion of the cell purge or during thebacterial fermentation process, such large amount of microbial biomasscan be useful for other applications. However, further complexprocessing is required to extract high quantities offermentation-derived proteins to high quality (i.e., with no harmfulsubstances or contaminants) for them to be useful, for example, as anutritious supplements or animal feedstock. Specifically, the presentinvention includes a process of extracting such fermentation-derivedproteins out of a cell biomass from a bacterial fermentation process.More specifically, the present invention includes systems for abacterial fermentation process to extract one or morefermentation-derived protein-containing portions out of cell mass ormicrobial biomass for processing into nutrient supplements and animalfeeds.

FIG. 1A is a flow chart of one example of a method 100 of producing aprotein-rich nutrient supplement from a bacterial fermentation system.The method 100 of bacterial fermentation of a gaseous substrates may beoperated under conditions which favor formation of hydrocarbonaceouscompounds, carbohydrates, specific proteins, specific amino acids,and/or other desired components, while maintaining desired fermentationproducts levels, such as alcohol productivity levels.

At step 110, a fermentation medium is added to a fermentation vessel tocarry out a bacterial fermentation process. In addition, one or moregaseous substrates are delivered into the fermentation vessel and befermented by a bacterial culture, such as a culture containing anaerobicbacteria. Initially, the liquid fermentation medium contained in thefermentation vessel may include various types of suitable bacterialculture medium, fermentation medium or liquid nutrient medium. Thenutrient medium includes one or more vitamins and several minerals in aneffective amount to permit growth of the microorganism used and/or tofavor specific products being generated.

A culture medium suitable for anaerobic bacterial growth suitable for afermentation process in producing one or more oxygenatedhydrocarbonaceous compounds such as various types of ethanol, butanol,acetic acid, etc., among others using syngas such as carbon monoxide andhydrogen gas or another other suitable substrate can be used. Oneexample of a suitable fermentation medium is described in U.S. Pat. No.7,285,402, which is incorporated herein by reference. Other examples ofsuitable medium are described in U.S. Ser. Nos. 61/650,098 and61/650,093, both of which are incorporated herein by reference.

In addition, the one or more gaseous substrates used in the bacterialfermentation process of the method 100 may include various synthesis gas(i.e., syngas), off-gases from a steel production process, off-gasesfrom an iron production process, off-gases from a coal productionprocess, or any other suitable gas sources from industrial productionplants. In one embodiment, the gaseous substrates used in the bacterialfermentation process include a carbon monoxide (CO)-containing gaseoussubstrate and/or additional gases such as hydrogen gases, carbon dioxide(CO2), nitrogen gas (N2), and combinations thereof.

In one example, the carbon monoxide-containing gaseous substrates may behigh volume carbon monoxide-containing industrial flue gases. In someaspects, a gas that includes carbon monoxide is derived fromcarbon-containing waste gases. Carbon-containing waste gases includeindustrial waste gases or the gasification of other municipal solid orliquid wastes. As such, such industrial processes represent effectiveprocesses for capturing carbon that would otherwise be exhausted intothe surrounding environment. Examples of industrial flue gases includegases produced during ferrous metal products manufacturing, non-ferrousproducts manufacturing, petroleum refining processes, gasification ofcoal, gasification of biomass, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing.

In one example, the carbon monoxide-containing syngas is introduced intothe fermentation vessel at varying rates dependent on the size and typeof fermentation vessel used. In one aspect, the syngas is introducedinto the gas inlet at a rate of about 10 to about 50 ft³/sec. In anotheraspect, syngas is introduced at a rate of about 25 to about 35 ft³/sec.The term “syngas” or “synthesis gas” includes, but is not limited to,synthesis gas in a gas mixture that is rich in carbon monoxide (CO) andhydrogen (H₂), such as a gas mixture produced from steam reforming ofnatural gas or hydrocarbons to produce hydrogen, the gasification ofcoal, or other gases produced in some types of waste-to-energygasification facilities. Syngas is combustible and is often used as afuel source or as an intermediate for the production of other chemicals.Syngas can be provided from any known source.

For example, syngas may be sourced from the gasification of carbonaceousmaterials. Gasification involves partial combustion of biomass in anenvironment where the oxygen supply is restricted. The resulting gasmainly includes carbon monoxide gas and hydrogen gas. Syngas contains atleast about 10 mole % carbon monoxide, or at least about 20 mole %, or10 to about 100 mole %, or 20 to about 100 mole %, 30 to about 90 mole %carbon monoxide, or about 40 to about 80 mole % carbon monoxide, orabout 50 to about 70 mole % carbon monoxide. The syngas will have acarbon monoxide/carbon dioxide molar ratio of at least about 0.75, or atleast 1.0, or at least about 1.5. Suitable gasification methods andapparatuses thereof are provided in U.S. patent application Ser. Nos.13/427,144, 13/427,193, and 13/427,247, as well as U.S. Pat. App. Nos.61/516,667, 61/516,704, and 61/516,646, all of which are incorporatedherein by reference.

Further, at step 110, a bacterial culture is inoculated into thefermentation vessel. The fermentation medium is sterilized to removeundesirable microorganisms and the bacterial fermentation vessel orfermentation bioreactor is inoculated with a chosen microorganism ormixed bacterial culture. In one aspect, the bacteria used in thebacterial culture is an anaerobic bacteria. Examples of the anaerobicbacteria used includes acetogenic bacteria, such as those of the genusClostridium, e.g., strains of Clostridium ljungdahlii, including thosedescribed in WO 2000/68407, EP 117309, U.S. Pat. Nos. 5,173,429,5,593,886 and 6,368,819, WO 1998/00558 and WO 2002/08438, strains ofClostridium autoethanogenum (DSM 10061 and DSM 19630 of DSMZ, Germany)including those described in WO 2007/117157 and WO 2009/151342 andClostridium ragsdalei (P11, ATCC BAA-622) and Alkalibaculum bacchi(CP11, ATCC BAA-1772) including those described respectively in U.S.Pat. No. 7,704,723 and “Biofuels and Bioproducts from Biomass-GeneratedSynthesis Gas”, Hasan Atiyeh, presented in Oklahoma EPSCoR Annual StateConference, Apr. 29, 2010 and Clostridium carboxidivorans (ATCCPTA-7827) described in U.S. Patent Application No. 2007/0276447. Each ofthese references is incorporated herein by reference. Other suitablebacteria include those of the genus Moorella, including Moorella sp.HUC22-1, and those of the genus Carboxydothermus. In one embodiment, amixed bacterial culture is used, wherein the mixed bacterial cultureincludes two or more bacterial microorganisms.

Useful bacteria to culture in this fermentation process of method 100include Acetogenium kivui, Acetoanaerobium noterae, Acetobacteriumwoodii, Alkalibaculum bacchi CP11 (ATCC BAA-1772), Blautia producta,Butyribacterium methylotrophicum, Caldanaerobacter subterraneous,Caldanaerobacter subterraneous pacificus, Carboxydothermushydrogenoformans, Clostridium aceticum, Clostridium acetobutylicum,Clostridium acetobutylicum P262 (DSM 19630 of DSMZ Germany), Clostridiumautoethanogenum (DSM 19630 of DSMZ Germany), Clostridium autoethanogenum(DSM 10061 of DSMZ Germany), Clostridium autoethanogenum (DSM 23693 ofDSMZ Germany), Clostridium autoethanogenum (DSM 24138 of DSMZ Germany),Clostridium carboxidivorans P7 (ATCC PTA-7827), Clostridium coskatii(ATCC PTA-10522), Clostridium drakei, Clostridium ljungdahlii PETC (ATCC49587), Clostridium ljungdahlii ERI2 (ATCC 55380), Clostridiumljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii O-52 (ATCC55889), Clostridium magnum, Clostridium pasteurianum (DSM 525 of DSMZGermany), Clostridium ragsdali P11 (ATCC BAA-622), Clostridiumscatologenes, Clostridium thermoaceticum, Clostridium ultunense,Desulfotomaculum kuznetsovii, Eubacterium limosum, Geobactersulfurreducens, Methanosarcina acetivorans, Methanosarcina barkeri,Morrella thermoacetica, Morrella thermoautotrophica, Oxobacterpfennigii, Peptostreptococcus productus, Ruminococcus productus,Thermoanaerobacter kivui, and combinations thereof. Other acetogenic oranaerobic bacteria may also be selected for use in the method 100described herein.

In one example, the bacteria used include acetogenic bacterial cellshaving a genomic DNA G+C content of about 50% or less. The acetogenicbacteria may be active, inactive or a combination of both. In thisaspect, G+C content may be determined by any methods known in the art.For example, the genome may be sequenced using methods such as thosedescribed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor) (alsoknown as “Maniatis”, which is incorporated herein by reference). G+Ccontent may then be determined manually or by using any number ofprograms, such as for example, Bohlin et al. “Analysis of IntragenomicGC Content Homogenicity within Prokaryotes”, BMC Genomics 2010, 11:464,which is incorporated herein by reference. Other methods for determiningG+C content include U.S. Pat. No. 8,143,037, Mesbah et al. (1989)“Measurement of Deoxyguanosine/Thymidine Ratios in Complex Mixtures byHigh-Performance Liquid Chromatorgraphy for Determination of the MolePercentage Guanine+Cytosine of DNA. J. Chromatogr. 479: 297-306, andTanner et al., “Costridium ljungdahlii sp. nov., an Acetogenic Speciesin Clostridial rRNA Homology Group I”, International Journal ofSystematic Bacteriology, April 1993, p. 232-236, all of which areincorporated herein by reference.

At step 110, upon inoculation of the bacterial culture into thefermentation vessel, an initial feed gas supply rate is established forthe effective growth of the initial population of the microorganisms(e.g., the anaerobic bacteria) and subsequent fermentation. Thefermentation vessel provides an environment to culture anaerobicbacteria. Suitable fermentation vessel may include, but is not limitedto, one or more of the following: a continuous stirred tank reactor(CSTR), an immobilized cell reactor (ICR), a trickle bed reactor (TBR),moving bed biofilm reactor (MBBR), a bubble column, a gas liftfermenter, a membrane reactor (e.g., a hollow fiber membrane bioreactor(HFMBR)), a static mixer, a vessel, a piping arrangement, a tower, aloop reactor, and combinations thereof. In the method 100, any knownfermentation vessels or fermentation bioreactors may be utilized. Someexamples of bioreactors are described in U.S. Ser. Nos. 61/571,654 and61/571,565, filed Jun. 30, 2011, U.S. Ser. No. 61/573,845, filed Sep.13, 2011, U.S. Ser. Nos. 13/471,827 and 13/471,858, filed May 15, 2012,and U.S. Ser. No. 13/473,167, filed May 16, 2012, all of which areincorporated herein by reference.

In one embodiment, the fermentation vessel includes a first bioreactorconnected to a second bioreactor, wherein the first bioreactor feeds afermentation liquid into the second bioreactor, wherein ethanolproduction takes place in the second bioreactor. For example, thefermentation vessel may be a two-stage CSTR system for improved culturestability. As an example, the fermentation vessel may optionally includea first Growth Stage with a first CSTR chamber and a second ProductionStage with a second CSTR chamber.

In one example, the Growth Stage CSTR is fed with a liquid culturemedium and unconverted substrate gas from the Production Stage CSTR isfed into the Growth Stage CSTR. In general, the Production Stage CSTR isfed with a fresh gas feed, and a fresh medium feed as well as abacterial culture feed from the Growth Stage CSTR. Optionally, cellrecycle is used to get the bacterial cells out of the Production StageCSTR, separated form fermentation products and sent back to theProduction Stage CSTR to obtain high bacterial fermentation efficiency.In general, bacterial cells are not recycled to the Growth Stage CSTR.U.S. patent Ser. No. 10/311,655 describes a continuous fermentationprocess and is herein incorporated by reference. The terms“fermentation”, fermentation process,” “bacterial fermentation process,”“fermentation reaction,” “bacterial fermentation reaction” and the likeare intended to encompass both the growth phase and product biosynthesisphase of the process. In one aspect, fermentation refers to conversionof carbon monoxide to alcohol. In one aspect, the bacterial fermentationprocess begins with the addition of a suitable fermentation medium andone or more gaseous substrates to the fermentation vessel containingbacteria therein.

In general, a fermentation liquid broth is generated inside thefermentation vessel once a bacterial fermentation process has started.The fermentation liquid broth may include one or more fermentationproducts, in addition to the culture medium, the one or more gaseoussubstrates, and the bacteria, contained inside the fermentation vessel.The fermentation products contained within the fermentation liquid brothand produced by the bacterial fermentation process inside thefermentation vessel may include one or more oxygenated hydrocarbonaceouscompounds, such as alcohols, etc., including, but not limited to,ethanol, 2-butanol, 2-butanone, 2,3-butanediol, acetone, butadiene,butane, butanol, butyrate, butyric acid, ethylene, and fatty acids,acetic acids, and combinations thereof.

In one aspect, a mixture of ethanol and acetic acid can be produced. Inanother aspect, the mixture of ethanol and butanol are produced. In oneexample, ethanol is produced in the fermentation vessel at a specificproductivity greater than 10 g/L per day, whereas free acetic acidconcentration is kept at less than 5 g/L of free acetic acid. Ethanoland acetate found in the fermentation liquid broth may be in a ratio ofethanol to acetate ranging from 1:1 to 20:1.

The fermentation liquid broth contained inside the fermentation vesselmay contain ethanol in diluted concentration and may need to be furtherprocessed in quality and/or its concentration. For example, thefermentation products contained in the fermentation liquid broth can bedelivered out of the fermentation vessel and into a distillation chamberor other types of reactors to be distilled into a final distillationproduction at higher concentration, and further processed and recovered.

The fermentation liquid broth may also include dead or inactivebacterial cells. These bacterial cells are otherwise known as bacterialcells or cells of anaerobic bacteria. An accumulation of cells from thebacterial fermentation process in large quantity is known as cell massor spent biomass. The term “inactive acetogenic bacteria” or “inactivebacterial cells” refers to dead cells which have lost their ability toreplicate after having gone through the bacterial fermentation process.The term “cell mass” refers to bacterial cells forming a microbialbiomass as a whole. The microbial biomass may accumulate duringbacterial fermentation and are useful to be processed by the methods andsystems described herein into fermentation-derived proteins. Thefermentation liquid broth may also include various proteins, aminoacids, carbohydrates, nucleic acids, and other moieties. Examples ofnucleic acid include nucleotides, such as DNA, RNA and any derivativesand analogs thereof. Due to the accumulation of cell mass or microbialbiomass in the fermentation broth, the fermentation broth itself mayprovide a significant caloric value. The fermentation liquid broth mayhave a dry matter content that is around 0.5%, 1%, 5%, 10%, 20%, 25%,30%, 35%, 40%, 45%, and around 50%.

As shown in FIG. 1A, at step 120 of the method 100, an amount of afermentation liquid broth containing cells of the anaerobic bacteria ata first concentration and the fermentation products are delivered out ofthe bacterial fermentation vessel. In general, when the cells reachedsteady-state growth inside the fermentation vessels, the fermentationliquid broth containing bacteria cells may be delivered out of thefermentation vessel. For a fermentation process after a steady-statebacterial growth stage, the first concentration of the first batch ofbacterial cells contained within the fermentation liquid broth may be0.5 g/L (dry cell mass) or higher, such 1.0 g/L or higher, or 2.0 g/L orhigher, or 5.0 g/L or higher, or 15.0 g/L or higher, or 30.0 g/L orhigher.

Next, at step 130, the cells of the anaerobic bacteria from thefermentation liquid broth are separated into a cell-free permeatesolution and a cell-containing suspension, using for example, one ormore cell separators. At this step, the goal is to separate and removethe bacterial cells from the fermentation liquid broth and obtain thecell-free permeate solution and the cell-containing suspensionseparately. The cell-free permeate solution contains mainly thefermentation products generated by the fermentation process and is readyfor further processing by distillation and other processes. Thecell-containing suspension is comprised mainly of bacterial cells afterthe fermentation process. The bacterial cells within the cell-containingsuspension can be measured at a second concentration (or second celldensity), and in one embodiment, the second concentration of thebacterial cells within the cell-containing suspension is equal to orhigher than the first concentration of the bacterial cells containedwithin the fermentation liquid broth.

To maintain a desired cell concentration of microbial culture in thefermentation vessel, the bacterial fermentation process includes purginga portion of the fermentation liquid broth. Increased cell concentrationgives rise to operation-related problems during fermentation, e.g., anunwanted increase in the concentration of free acetic acid, such thatthe production of acetate becomes favored over the production ofethanol. Thus, it is important to monitor cell density and conductperiodic or continuous cell purges of the fermentation liquid broth. Theterm “cell density” means mass of microorganism cells per unit volume offermentation medium, for example, grams/liter.

Stabilization of cell concentration in the bacterial fermentation vesselis accomplished by purging bacterial cells from the fermentation vesselto a cell concentration less than the stable steady state concentrationthat utilizes all reducing gas or nutrient substrates in the bioreactorand increasing the aqueous feed rate when the free acetic acid portionof the acetate present in the fermentation bioreactor broth exceeds ahigh concentration (e.g., a free acetic acid concentration of 1 g/L orhigher, or 2 g/L or higher). Large scale, continuous bacterialfermentation can be maintained for a long time (e.g., for many months)by maintaining a constant cell concentration within the fermentationvessel without additional culture supplementation. Bacterial culturewithin the fermentation vessel is fed one or more gases (e.g., CO, CO₂,H₂, and other carbon source substrates) along with a liquid nutrientmedium containing vitamins and other essential nutrients during thisperiod.

Suitable cell separators that can be used to separate the cell-freepermeate solution from the cell-containing suspension within thefermentation liquid broth include, but are not limited to, anyfiltration devices, hollow fiber filtration devices, spiral woundfiltration devices, ultrafiltration devices, ceramic filter devices,cross-flow filtration devices, size exclusion column filtration devices,or combinations thereof. Suitable filters that can be used in thefiltration-type cell separators of the invention include, but are notlimited to, spiral wound membranes/filters, cross flow filters. Inaddition, another suitable means of cell separation from cell-freepermeate is through the use of one or more centrifugation devices.

In one embodiment, the cell-separator used at step 130 functions toseparate bacterial cells into the cell-containing suspension and thecell-free permeate solution and/or concentrate the cell-containingsuspension to be at a higher concentration than the cell concentrationwithin the fermentation liquid broth prior to cell separation by thecell separator. In an alternative embodiment, the cells within thefermentation liquid broth can be separated and concentrated by sendingit several passes through the cell separator (e.g., by several passesthrough one or more filter-type filtration devices or centrifugations byone or more centrifuge several times at the same or differentcentrifugation speeds).

In a preferred embodiment, after cell separation, the cell concentration(or cell density) within the cell-containing suspension is higher thanthe cell concentration of the fermentation liquid broth. In one aspectof the invention, one or more filtration devices with spiral woundfilters are used to concentrate cells by sending the fermentation liquidbroth through the spiral wound filters several passes.

In another aspect, cell recycle is performed, and is generally referredto as the separation of a bacterial cell containing suspension from acell-free liquid permeate solution and returning all or part of thoseseparated bacterial cells back to the fermentation vessel. In oneembodiment, ultrafiltration by a cell separator, such as a filtrationdevice, is used to accomplish cell separation and/or cell recycle.

In still another aspect, during steady state bacterial growth for thebacteria cultured within the fermentation vessel, a cell purge from thefermentation vessel is conducted to collect bacterial cells into higherconcentrations of the cell-containing suspension or semi-dry microbialbiomass. In one embodiment, a cell purge requires an amount offermentation liquid broth containing bacterial cells and othersubstances found in a fermentation medium. For example, the cell purgemay be a fermentation or fermentation liquid broth removed from thefermentation vessel during bacterial fermentation. In anotherembodiment, the cell purge may require obtaining a concentratedcell-containing suspension by removing the fermentation liquid at afirst cell concentration from the fermentation vessel and furtherconcentrating the cells to have a cell-containing suspension at a secondcell concentration. The cell-containing suspension has a higher celldensity than the cell density of the fermentation liquid removed fromthe fermentation vessel. These steps provide for the efficient removalof certain particulates and allows for a high yield of protein contentin the final protein-rich nutrient supplement that is produced from themethod 100.

In one aspect, the cell purge occurs during a continuous bacterialfermentation. In another aspect, the cell purge occurs after bacterialfermentation, wherein the bacterial fermentation process is paused orstopped to permit the removal of microbial biomass from the fermentationvessel.

At step 140 of the method 100, the bacterial cells contained within thecell-containing suspension are ruptured into a homogenate. A rupturingdevice can be used to rupture and/or lyse the cell membranes ofbacterial cells within the cell-containing suspension. Examples of therupturing device for rupturing bacterial cells include, but are notlimited to, various types of microfluidics devices, sonication devices,ultrasonic devices, mechanical disruption devices, French press,freezers, heaters, high temperature reactors, homogenizers, andcombinations thereof, among others.

As shown in FIG. 1, after the bacterial cells within the cell-containingsuspension are broken-open and/or ruptured, the resulting ruptured cellmixture, e.g., a homogenate, can be further processed at step 150 byseparating out a protein-containing portion from a cell debris portionwithin the homogenate of the microbial biomass and further purifying andextracting additional protein-containing portions to generate aprotein-rich nutrient supplement. Such separation is contemplated to beperformed by the use of one or more fractionators. In one aspect, one ormore protein-containing portions are obtained and the one or moreprotein may also include free amino acids, total amino acids, andpeptides.

At Step 150, suitable examples of the one or more fractionators forfractioning the homogenate include, but are not limited to, varioustypes of solid-liquid fractionators, centrifugation devices, continuouscentrifuges, decanter centrifuges, disc-stack centrifuges, a filtrationdevices, a hollow fiber filtration device, a spiral wound filtrationdevice, a ceramic filter device, a cross-flow filtration device, a sizeexclusion device, one or series of size exclusion columns, one or seriesion exchange columns, one or series of carbon polymer columns, aflow-through magnetic fractionator, an ultrafiltration device, one orseries of affinity chromatography columns, one or series of gelfiltration columns, and combinations thereof, among others.

In one embodiment, at step 150, the homogenate obtained after rupturingof bacterial cells by one or more rupturing devices is delivered to afirst fractionator, and a first protein-containing portion and a firstprotein-containing cell debris portion are obtained. In one aspect, thefirst protein-containing portion has a protein content of at least 1% ormore, 3% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40%or more, 50% or more, 60% or more, 70% or more, 80% or more, or 95% ormore.

In one embodiment, at step 160, the first protein-containing portionderived from the homogenate can be directly incorporated intoprotein-rich nutrient supplement compositions, cell growth mediumsupplement/composition, pharmaceutical compositions, and/or an animalfeed (e.g., fish feed, shrimp feed, feed for chicken, etc.). Suchincorporation may require drying of the first protein-containing portioninto low moisture content (e.g., paste or powder forms) and directblending of the first protein-containing portion with other ingredients(e.g., additional animal feed nutrients, pharmaceutical fillers,blending agent, plasticizers, etc.) for making one or more types ofnutrient supplements. The step 160 described herein may includeadditional processing steps of adjusting the pH of the firstprotein-containing portion, addition of one or more solubilityenhancers, removal of harmful proteins from the first protein-containingportion, and/or combinations thereof to increase and enhance the qualityand concentration of first protein-containing portion. In addition, thefirst protein-containing portion may undergo further downstreamprocessing by performing extraction and purification of bacterialfermentation-derived protein and repurposing it for use as aprotein-rich nutrient supplement. Such examples are shown in in FIGS. 2Aand 2B.

Alternatively, at step 165, the first protein-containing cell debrisportion derived from the homogenate can be directly incorporated intoprotein-rich nutrient supplement compositions, pharmaceuticalcompositions, cell growth medium supplement/composition, and/or ananimal feed (e.g., fish feed, shrimp feed, feed for chicken, etc.).Similarly, additional processing steps of adjusting the pH of the firstprotein-containing cell debris portion, addition of one or moresolubility enhancers to the protein-containing cell debris portion,removal of harmful proteins from the first protein-containing celldebris portion, and/or combinations thereof may be needed to increaseand enhance the quality and concentration of first protein-containingcell debris portion. In one aspect, if the soluble proteins of the firstprotein-containing cell debris portion alone are recovered, then therecovered proteins can be obtained and directly incorporated as anutrient-rich supplement for animal intake or human intake. However, forthe first protein-containing cell debris portion to be incorporated intohigh quality nutrient-rich supplements for human intake, furtherdownstream processing to purify and recover nutrients and proteincontents may be required. In another aspect, the insoluble proteinsrecovered in this method can undergo further downstream processing andthen be combined with the first protein-containing portion and producedas a protein-rich nutrient supplement.

FIG. 1B is a flow chart of another example of a method 100 of producinga protein-rich nutrient supplement from a bacterial fermentation system.The method 100 of bacterial fermentation of a gaseous substrates may beoperated under conditions which favor formation of hydrocarbonaceouscompounds, carbohydrates, specific proteins, specific amino acids,and/or other desired components, while maintaining desired fermentationproducts levels, such as alcohol productivity levels.

Step 130 of the method 100 includes separating the cells of theanaerobic bacteria from the fermentation liquid into a cell-freepermeate solution and a cell-containing suspension containing anaerobicbacterial cells at a second cell concentration. Optionally, step 135 ofthe method 100 includes holding the cell-containing suspension in afirst holding tank. In one aspect, the bacterial cells can undergopretreatment in preparation of generating a protein-rich supplement withhigh protein content and appropriate for consumption. In still anotheraspect, one or more additives added in the pre-treatment process at step130 may also help to optimize the conditions for rupturing the bacterialcells and generate the homogenate at high quality.

Suitable additives to be used at step 130 include, but are not limitedto, detergents, pH-adjusting agents, enzymes, nuclease, protease,hydrolases, alkaline buffer, acidic buffer, or combinations thereof. Inone embodiment, the step 130 of the method 100 includes reducing thenucleic acid content of the cell-containing suspension offermentation-derived bacterial cells. Such pretreatment process isaccomplished by treating the bacterial cells with nucleases. Examples ofnucleases used include, but are not limited to, deoxyribonucleases,ribonucleases, benzonases, and nuclease. Nuclease treatment of thecell-containing suspension can be further assisted by alkalinehydrolysis and chemical extraction, such as ammonium sulfateprecipitation, ethanol precipitation, polyethyleneimine precipitation.

Step 140 of the method 100 includes rupturing cell membranes of theanaerobic bacterial cells within the cell-containing suspension togenerate a homogenate by a rupturing device. Optionally, step 145 of themethod 100 includes holding the cell-containing suspension after beingruptured in a first holding tank. In one aspect, the bacterial cells canundergo process in preparation of generating a protein-rich supplementwith high protein content and appropriate for consumption. In stillanother aspect, one or more additives added in the process at step 145may also help to optimize the conditions after rupturing the bacterialcells.

Suitable additives to be used at step 145 include, but are not limitedto, detergents, pH-adjusting agents, enzymes, nuclease, protease,hydrolases, alkaline buffer, acidic buffer, or combinations thereof. Inone embodiment, the step 145 of the method 100 includes reducing thenucleic acid content of the homogenate of fermentation-derived bacterialcells. Such pretreatment process is accomplished by treating thebacterial cells with nucleases. Examples of nucleases used include, butare not limited to, deoxyribonucleases, ribonucleases, benzonases, andnuclease. Nuclease treatment of the cell-containing suspension can befurther assisted by alkaline hydrolysis and chemical extraction, such asammonium sulfate precipitation, ethanol precipitation, polyethyleneimineprecipitation.

Step 150 of the method 100 includes fractionating the homogenate into afirst protein-containing portion and a protein-containing cell debrisportion using a first fractionator. Step 160 of the method 100 includesobtaining the first protein-containing portion as a protein-richnutrient supplement. Step 165 of the method 100 includes obtaining theprotein-containing cell debris portion as a protein-rich nutrientsupplement.

FIG. 2A is one example of a method 200A of processing a cell-containingsuspension containing anaerobic bacterial cells from a fermentationprocess (e.g., a cell-containing suspension at a second concentrationfrom the step 130 of the method 100) to obtain a secondprotein-containing portion as a protein-rich nutrient supplement. In themethod, there is an optional processing step of treating thecell-containing suspension with one or more additives at step 202 priorto rupturing the cell membranes of the aerobic cells withincell-containing suspension and generating the homogenate.

In the cell pre-treatment process of step 202, the cell-containingsuspension can be processed in a pre-treatment chamber or a holding tankfor pre-treatment and treated with one or more additives to assist andincrease cell rupturing efficiency to break down the cell walls and cellmembranes of the anaerobic bacterial cells at step 204. In one aspect,the concentrated bacterial cells enter a holding tank where they arehoused until the rupturing device is ready for larger volume of cells tobe processed together. In another aspect, while housed in the holdingtank, the bacterial cells can undergo pretreatment in preparation ofgenerating a protein-rich supplement with high protein content andappropriate for consumption. In still another aspect, one or moreadditives added in the pre-treatment process at step 202 may also helpto optimize the conditions for rupturing the bacterial cells at step 204and generate the homogenate at high quality.

Suitable additives to be used at step 202 include, but are not limitedto, detergents, pH-adjusting agents, enzymes, nuclease, protease,hydrolases, alkaline buffer, acidic buffer, or combinations thereof. Inone embodiment, the step 202 of the method 200A includes reducing thenucleic acid content of the cell-containing suspension offermentation-derived bacterial cells. Such pretreatment process isaccomplished by treating the bacterial cells with nucleases. Examples ofnucleases used include, but are not limited to, deoxyribonucleases,ribonucleases, benzonases, and nuclease. Nuclease treatment of thecell-containing suspension can be further assisted by alkalinehydrolysis and chemical extraction, such as ammonium sulfateprecipitation, ethanol precipitation, polyethyleneimine precipitation.In one aspect, the nucleic acid content of the cell-containingsuspension is reduced to about 1.5% to 5%, or about 2% to 18%.

At step 206, after cell rupturing, the homogenate can be subjected toadditional extraction and purification processes, such as beingfractionated into the first protein-containing portion and aprotein-containing cell debris portion using a first fractionator.Example of the first fractionator for fractioning the homogenateincludes, but are not limited to, various types of solid-liquidfractionators, centrifugation devices, continuous centrifuges, decantercentrifuges, disc-stack centrifuges, a filtration devices, a hollowfiber filtration device, a spiral wound filtration device, a ceramicfilter device, a cross-flow filtration device, a size exclusion device,one or series of size exclusion columns, one or series ion exchangecolumns, one or series of carbon polymer columns, a flow-throughmagnetic fractionator, an ultrafiltration device, one or series ofaffinity chromatography columns, one or series of gel filtrationcolumns, and combinations thereof, among others.

Alternatively, the cell-containing suspension of concentrated bacterialcells may go directly to a rupturing device at step 204 and are thensubjected to pre-treatment process as mentioned at step 202 to helpoptimize the conditions for separating and fractionating the homogenateat step 206. In another aspect, the proteins recovered from the method200A can undergo further downstream processing and then be combined withthe first protein-containing portion and produced as a protein-richnutrient supplement.

At step 208, the first protein-containing portion is delivered to asecond fractionator and fractionated into a second protein-containingportion using the second fractionator at step 210. Examples of thesecond fractionator for fractioning the protein-containing portionincludes, but are not limited to, various types of solid-liquidfractionators, centrifugation devices, continuous centrifuges, decantercentrifuges, disc-stack centrifuges, a filtration devices, a hollowfiber filtration device, a spiral wound filtration device, a ceramicfilter device, a cross-flow filtration device, a size exclusion device,one or series of size exclusion columns, one or series ion exchangecolumns, one or series of carbon polymer columns, a flow-throughmagnetic fractionator, an ultrafiltration device, one or series ofaffinity chromatography columns, one or series of gel filtrationcolumns, and combinations thereof, among others.

At step 212, the second protein-containing portion obtained can beformulated into a protein-rich nutrient supplement, pharmaceuticalcompositions, cell growth medium/composition, and/or an animal feed(e.g., fish feed, shrimp feed, feed for chicken, etc.). Suchincorporation may require drying of the second protein-containingportion into low moisture content (e.g., paste or powder forms) anddirect blending of the second protein-containing portion with otheringredients (e.g., additional animal feed nutrients, pharmaceuticalfillers, blending agent, plasticizers, etc.) for making one or more typeof nutrient supplements.

As an example, the homogenate from step 204 may enter a filtrationdevice to yield a first protein-containing portion (e.g., the filtrateprotein-containing portion after filtration by the filtration device).The filtrated protein-containing portion is a partially purified proteinproduct. In one embodiment, the filtrated protein-containing portion isthen centrifuged (e.g., by a second fractionator/centrifuge) to yieldadditional soluble protein fractions and cell solids portions. Suchsecond protein-containing portions can be used individually or incombination as the protein-rich nutrient supplements.

As another example, the homogenate from step 204 may undergocentrifugation, after which a supernatant protein-containing portion iscollected. The supernatant protein-containing portion enters afiltration device, wherein a second filtrate protein-containing portionis collected. As yet another example, the first protein-containingportion only enters a filtration device, after which a filtrateprotein-containing portion is collected and used as a protein-richsupplement. As yet another example, the first protein-containing portiononly undergoes centrifugation, after which a protein fractionate orsupernatant protein-containing portion is collected. Separation of oneor more protein-containing portions and cell-debris proteins isaccomplished by one or more fractionators.

In one aspect, a fermentation system that has two fractionators is used.In another embodiment, a fermentation system that has threefractionators is used. FIG. 2B is one example of a method 200B ofprocessing a cell-containing suspension from a fermentation processwhere three fractionators are used to obtain a third protein-containingportion as a protein-rich nutrient supplement according to one or moreembodiments of the invention.

As shown in FIG. 2B, the method 200B includes delivering the secondprotein-containing portion to a third fractionator at step 214 and athird protein-containing portion is fractionated from and extracted outof the second protein-containing portion at step 216. The thirdprotein-containing portion is collected at step 218 and the thirdprotein-containing portion is collected and obtained as a protein-richsupplement at step 220.

As an example, the second protein-containing portion from step 210 maybe delivered to enter a third fractionator (e.g., a filtration device)to yield a filtrate protein-containing portion collected and used as aprotein-rich supplement. As another example, the secondprotein-containing portion undergoes centrifugation, after which asupernatant protein-containing portion and a pellet cell debris portionare collected. The filtrate protein-containing portion is collected fromthe filtration device and produced as the protein-rich nutrientsupplement with a protein content of around 1 to 3%, 3% to 7%, 7% to10%, around 10% to 14%, around 11% to 20%, around 21% to 35%, and around35% or more.

In one embodiment, one or more protein-containing portions obtained fromsteps 160, 165, 212, 220 are delivered to a dehydration chamber, afterwhich a dehydrated protein-containing portion is collected and producedas a protein-rich nutrient supplement. Alternatively, there are two ormore dehydration chambers, wherein each protein-containing portion fromeach step is delivered to a separate, individual dehydration chamber.The dehydration chamber receives the protein-containing portions anddries them into low moisture paste forms or dry power forms, ready to beblended into protein-rich nutrient supplements for human intake and/oranimal feeds. Suitable examples of the dehydration chambers include, butare not limited to, an oven dryer, a spray drying chamber, a drum dryer,and a freeze dryer, a lyophilization device, and combinations thereof.

In one embodiment, the present invention is a method of producing aprotein-rich nutrient supplement from a fermentation process usinganaerobic bacteria. The method includes fermenting a gaseous substratewith anaerobic bacteria in a fermentation vessel, obtaining from thefermentation vessel an amount of a fermentation liquid containing cellsof the anaerobic bacteria at a first concentration, separating the cellsof the anaerobic bacteria from the fermentation liquid into a cell-freepermeate solution and a cell-containing suspension containing anaerobicbacterial cells at a second concentration, rupturing cell membranes ofthe anaerobic bacterial cells within the cell-containing suspension intoa homogenate, and separating a first protein-containing portion from acell debris portion within the homogenate.

In one aspect, the method includes fermenting a gaseous substrate withanaerobic bacteria in a fermentation vessel. The gaseous substrate is aCO-containing gaseous substrate of one or more gases that flows into thefermentation vessel. The one or more gases used is selected from thegroup consisting of carbon source substrates, carbon monoxide (CO),carbon dioxide (CO₂), hydrogen (H₂) gas, syngas, and combinationsthereof. Anaerobic bacteria include, but are not limited to, one or morestrains of acetogenic bacteria, such as from the genus Clostridium,Acetobacterium, and similar variants thereof. The fermentation vesselprovides an environment that is hospitable for culturing Clostridiumbacteria, wherein there is a fermentation medium that flows into thefermentation vessel to provide nutrients, vitamins, and other essentialminerals to the bacteria.

The method further includes obtaining from the fermentation vessel anamount of a fermentation liquid containing cells of the anaerobicbacteria at a first cell concentration. Collections of the fermentationliquid may be sent to one or more apparatuses within the bacterialfermentation system. In one aspect, subsequent manipulated amounts offermentation liquid are at a second, third, and fourth cellconcentration. In most aspects, the second cell concentration of amanipulated fermentation liquid is greater than the first cellconcentration of a first fermentation liquid.

The method further includes a first cell separator receiving an amountof fermentation liquid that contains anaerobic bacterial cells. Thefirst cell separator separates the fermentation liquid into a firstcell-containing suspension containing anaerobic bacterial cells and afirst cell-free permeate solution. The fermentation liquid delivered tofirst cell separator has a first cell concentration. The firstcell-containing suspension generated by the first cell separator has asecond cell concentration. The second cell concentration of the firstcell-containing suspension is higher than the first cell concentrationof the fermentation liquid. The first cell-free permeate solution issent to a processing chamber connected to the first cell separator. Insome aspects, some of the first cell-containing suspension is sent backto the fermentation vessel.

In another aspect, a first flow of the first fermentation liquid is sentto a first cell separator to further process for ethanol production. Asecond flow of the second fermentation liquid is sent to a second cellseparator to further process for the production of a protein-containingproduct that can be used as a protein-rich nutrient supplement.

The method of the present invention provides a simultaneous approach ofgenerating a high productivity of ethanol production, while re-purposinguseful moieties found within bacterial cells used in the fermentationprocess. The fermentation liquid collected is at a first concentrationof anaerobic bacterial cells. The cell separator generates acell-containing suspension at a second concentration of anaerobic cells.In one embodiment, the cell separator sends the cell-containingsuspension at a second concentration to a rupturing device. In anotherembodiment, the cell separator sends the cell-containing suspension to aholding tank.

Once a collection is made, the collection can be further processed toseparate the cells of the anaerobic bacteria from the fermentationliquid into a cell-free permeate solution and a cell-containingsuspension containing the anaerobic bacterial cells at a secondconcentration. The second concentration of the cell-containingsuspension is higher than the first concentration of fermentation liquidcontaining anaerobic bacterial cells. The cell-free permeate solution issent back to a processing chamber that distills ethanol for ethanolproduction. This provides for an efficient system that does not discardstill useful cell-free permeate solution containing ethanol.

The method further includes rupturing cell membranes of the anaerobicbacterial cells within the cell-containing suspension into a homogenate.In one aspect, this takes place in a rupturing device. Thecell-containing suspension containing cells of anaerobic bacteria entersthe rupturing device, wherein the cell-containing suspension issubjected to high forces (e.g., mechanical, sound, or pressure). Thehigh shear force ruptures the cell membranes of the cells, causing thecells to break open and for the contents of the cells to befree-floating as they enter the cell-containing suspension. Therupturing device generates a homogenate that can be further processed toobtain a first protein-containing portion. The homogenate containsseveral moieties generally found in fermentation-derived bacterialcells, including proteins, metals (e.g., Ca, Cl, Co, K Mg, Ni, P, S, Se,W, Zn, Na, Fe), lipids, nucleic acids, and sugars.

To obtain a first protein-containing portion, the method furtherincludes separating a first protein-containing portion from a celldebris portion within the homogenate. In one aspect, the homogenate iscentrifuged, and then filtered, to yield a first protein-containingportion. The first protein-containing portion is delivered to a firstfractionator, which separates a second protein-containing portion fromthe first protein-containing portion and allows collection of a secondprotein-containing portion from the first fractionator.

In one aspect, the method includes dehydrating the firstprotein-containing portion separated from the cell debris portion of thehomogenate. In this aspect, the system has a dehydration chamberconnected to a first fractionator. The first fractionator delivers thefirst protein-containing portion into the dehydration chamber, whereinthe dehydration chamber generates a dried protein-containing portionproduced as a protein-rich nutrient supplement.

In another aspect, the method includes dehydrating the cell debrisportion of the homogenate. In this aspect, the rupturing device deliversthe cell debris portion into a dehydration chamber, to be prepared forfurther downstream processing. The cell debris portion is an insolublefraction containing a high level of protein content. Typically, thisincludes cell wall or cell membrane components that are insoluble. Itmay also contain small concentrations of nucleic acid or proteinaggregates. In most aspects, the majority of the nucleic acid isreleased into the first protein-containing portion. Sometimes theseprotein aggregates are difficult to solubilize and will remain in thecell debris portion. Determinations of protein content in the firstprotein-containing portion and the cell debris portion is based on anassumption of mass balance around the total cell mass and the proteinamounts, soluble and insoluble. By way of example, a calculation ofinsoluble protein recovery includes subtracting the mass of the solubleprotein from the total cell mass to yield an approximation of theinsoluble mass.

II. Bacterial Fermentation Systems for Processing an Acetogenic Biomassto Yield a Fermentation-Derived Protein

The bacterial fermentation system includes, but is not limited to, abacterial fermentation vessel, one or more rupturing devices, one ormore cell separators, and one or more fractionators. In addition, one ormore dehydration chambers are connected to the one or more rupturingdevices and/or the one or more fractionators to increase theprotein-concentration of the protein-containing portions obtained andreduce their moisture content. Optionally, the bacterial fermentationsystem further includes one or more holding tanks, storage chambers,and/or pre-treatment chambers for holding bacterial cells orcell-containing suspensions.

FIGS. 3A-3B, 4A-4H, 5A-5E, and 6 illustrate such exemplary bacterialfermentation systems for producing a cell-containing suspension and oneor more oxygenated hydrocarbonaceous compounds from a fermentationprocess using a culture of an anaerobic bacteria. FIG. 3A is a schematicof a bacterial fermentation system 300A for producing a cell-containingsuspension and one or more oxygenated hydrocarbonaceous compounds, wheretwo cell separators and one dehydration chamber are used.

In FIG. 3A, the bacterial fermentation system 300A includes afermentation vessel 310, a first cell separator 320, a second cellseparator 330, a processing chamber 350, and optionally, a dehydrationchamber 375. The bacterial fermentation system 300A can be, in oneembodiment, a continuous bacterial fermentation system. Alternatively,the bacterial fermentation system 300A can be, a batch bacterialfermentation system.

Two or more inlet lines, e.g., an inlet line 302 and an inlet line 304,are connected to the fermentation vessel 310. The inlet line 302 can beused for delivery of gaseous substrates, additional supplements, and/orother solid or liquid substrates into the fermentation vessel 310. Theinlet line 304 can be used for delivery of a fermentation medium orother culture medium into the fermentation vessel 310. Conversion of thegaseous substrates and the fermentation medium takes place in thefermentation vessel 310. The fermentation medium used herein includesconventional bacterial growth media containing vitamins, salts, andminerals sufficient to permit growth of selected anaerobic bacteria.Vitamins in the form of a vitamin cocktail are added into thefermentation medium. Vitamins include several from the B vitamin family,including, but not limited to, thiamine (B1), pantothenic acid (B5),biotin (B7), other amino acids and combinations thereof.

Inside the fermentation vessel 310, the gaseous substrates and thefermentation medium are fermented by the anaerobic bacteria containedwithin the fermentation vessel 310 into a fermentation liquid broth,containing cells of the anaerobic bacteria at a first concentration. Thereactor gas is then released from the bacterial fermentation system 300Aby the outlet line 314. The fermentation vessel 310 provides anenvironment to ferment the gaseous substrate with anaerobic bacteria. Inone aspect, the gaseous substrate is one or more gases consisting ofcarbon source substrates, carbon monoxide (CO), carbon dioxide (CO₂),hydrogen gas (H₂), and syngas, whereas the anaerobic bacteria is one ormore anaerobic bacterium selected from the genus Clostridium,Acetobacterium, and variants thereof.

The fermentation vessel 310 may include three or more outlet lines,e.g., an outlet line 314, an outlet line 316, and an outlet line 312.The outlet line 314 can be used for delivery of gases, vent gases, extragases to be exhausted out of the fermentation vessel 310. The outletline 312 can be used for delivery of a portion of the fermentationliquid broth out of the bacterial fermentation system 300A to the firstcell separator 320. The outlet line 316 can be used for delivery of aportion of the fermentation liquid broth out of the bacterialfermentation system 300A to the second cell separator 330. Portions ofthe fermentation liquid broth from the fermentation vessel 310 aredelivered and supplied to the first cell separator 320 and second cellseparator 330, each by outlet line 312 and outlet line 316,respectively. Inside each of the first cell separator 320 and secondcell separator 330, the cells of the anaerobic bacteria contained withinthe fermentation liquid broth (containing bacterial cells at a firstconcentration) are separated into a cell-free permeate solution and aretentate solution (e.g., a cell-containing suspension containinganaerobic bacteria cells at a second concentration).

An outlet line 322 and an outlet 332 are used to deliver cell-freepermeate solutions out of the first cell separator 320 and a second cellseparator 330, respectively, and into the processing chamber 350. Insidethe processing chamber 350, the cell-free permeate solution is processedinto an oxygenated hydrocarbonaceous compound. The processing chamber350 may also recycle distillation aqueous contents, including water,back to the fermentation vessel 310 through an outlet line 354. In oneexample, the distillate may mainly include water, and may also containother contents. For example, general distillation aqueous streamcontains 95% of water, about 5% of acetic acid, and some other contents.Then, the processing chamber 350 sends out a final product of anoxygenated hydrocarbonaceous compound through an outlet line 352 forfurther downstream processing. In one embodiment, the processing chamber350 is a distillation chamber where cell-free permeate solution isprocessed and distilled into high quality oxygenated hydrocarbonaceouscompound (e.g., high concentration and/or anhydrous form of ethanol,butanol, such as 95% w/w or higher concentration of ethanol, etc.

The cell-containing suspension obtained after passing through the firstcell separator 320 can be delivery via an outlet line 324 back to thefermentation vessel 310 for cell recycle so that the cells within thecell-containing suspension may undergo further fermentation process. Onthe other hand, the cell-containing suspension obtained after passingthrough the second cell separator 330 can be concentrated by the secondcell separator 330 and delivered via an outlet line 336 to thedehydration chamber 375 to be ruptured into a mixture and dried. Thedehydration chamber 375 can be an oven dryer, a paddle dryer, a spraydrying device, a drum dryer, a lyophilization device, and combinationsthereof. A portion of the cell-containing suspension, containinganaerobic bacteria cells, in the second cell separator 330 is thendelivered back to the fermentation vessel 310 via an outlet line 334 forfurther fermentation process.

One example of processing of the cell-containing suspension into aprotein-rich supplement is to subject the cell-containing suspension ata high temperature of about 100 degree Celsius or higher (e.g., at 250degree Celsius or higher) inside a high temperature processing chamber,e.g., the spray drying dehydration chamber, to rupture the cells andreduce moisture content of the cell-containing suspension into paste orpowder forms. Another example of processing of the cell-containingsuspension is to subject the cell-containing suspension at a temperatureof about 0 degree Celsius or lower inside a low temperature processingchamber.

An outlet line 376 is connected to the dehydration chamber 375 todeliver the ruptured and dehydrated form of the cell-containingsuspension out of the dehydration chamber 375 to be ready for blendinginto compositions of protein-rich supplements. After the dehydrationprocess is undergone in the dehydration chamber 375, a protein-richnutrient supplement is obtained and collected from the bacterialfermentation system 300A via the outlet line 376.

FIG. 3B is a schematic of a bacterial fermentation system 300B forproducing a cell-containing suspension and one or more oxygenatedhydrocarbonaceous compounds, where two cell separators, one holding tankand one dehydration chamber are used. In FIG. 3B, the bacterialfermentation system 300B includes the fermentation vessel 310 connectedto the inlet line 302, the inlet line 304 and several outlet lines, thefirst cell separator 320 connected to the outlet line 312, the outletline 322, and the outlet line 324, the second cell separator 330connected to the outlet line 316 and the outlet line 336, and thedehydration chamber 375 connected to the outlet lines 336 and the outletline 376, as discussed above.

In addition, the bacterial fermentation system 300B further includes aholding tank or a storage tank for holding and storing portions of thecell-free permeate solutions. For example, a holding tank 340 (e.g., acell-free permeate holding tank) is connected to the first cellseparator 320 and the second cell separator 330 via the outlet line 322and an outlet line 332, respectively. The cell-free permeate solutionsobtained after cell separation by the first cell separator 320 and thesecond cell separator 330 can undergo pretreatment or storage into largequantity in preparation for processing the cell-free permeate solutionsbeing held in the holding tank 340 into a final product of high-qualityform of an oxygenated hydrocarbonaceous compound. In one embodiment, thecell-free permeate solution is further processed for ethanol productionwithin a processing chamber 350 being delivered from holding tank 340via an outlet line 342. Afterward, processed final products ofoxygenated hydrocarbonaceous compounds are delivered out of theprocessing chamber 350 via the outlet line 352, and water, acetic acid,nutrients, and other materials produced from the processing chamber 350can be recycled back to the fermentation vessel 310 via an outlet line354.

FIG. 4A shows a schematic of a bacterial fermentation system 400A withone fermentation vessel, one cell separator, one processing chamber, onerupturing device, one fractionator, and two dehydration chambers for afermentation process using a culture of an anaerobic bacteria accordingto one or more embodiments of the invention and obtaining a protein-richnutrient supplement from bacterial fermentation. The bacterialfermentation system 400A includes an inlet line 402, an inlet line 404,a fermentation vessel 410, an outlet line 412, an outlet line 414, acell separator 420, an outlet line 422, an outlet line 424, a processingchamber 450, an outlet line 452, an outlet line 454, a rupturing device460, an outlet line 462, a fractionator 470 an outlet line 472, anoutlet line 474, a dehydration chamber 475A, a dehydration chamber 475B,an outlet line 476A, and an outlet line 476B.

The bacterial fermentation system 400A can be, in one embodiment, acontinuous bacterial fermentation system. First, a flow of fermentationmedium is supplied to the bacterial fermentation system 400A by theinlet line 402. And a flow of gaseous substrates is supplied to thebacterial fermentation system 400A by the inlet line 404. The flow ofgaseous substrates and the fermentation medium then enter into thefermentation vessel 410 that cultures anaerobic bacteria. Thefermentation vessel 410 provides an environment to ferment the gaseoussubstrate with anaerobic bacteria. Conversion of the gaseous substratesand the fermentation medium takes place in the fermentation vessel 410.Inside the fermentation vessel 410, the gaseous substrates and thefermentation medium are fermented facilitated by the anaerobic bacteriacontained within the fermentation vessel into a fermentation liquidbroth, containing cells of the anaerobic bacteria at a firstconcentration. Unreacted reactant gases are then released and exhaustedfrom the bacterial vessel 410 by the outlet line 414.

Further, the fermentation liquid broth is delivered and supplied to thefirst cell separator 420 by outlet line 412. Inside the first cellseparator 420, the cells of the anaerobic bacteria contained within thefermentation liquid broth are separated into a cell-free permeatesolution and a cell-containing anaerobic bacteria cells at a secondconcentration. The cell-free permeate solution in the first cellseparator 420 is then delivered to the processing chamber 450 via theoutlet line 422. An amount of the cell-containing suspension, containinganaerobic bacteria cells, in the first cell separator 420 is thendelivered back to the fermentation vessel 410 via the outlet line 424 toundergo further fermentation process. Another amount of thecell-containing suspension, containing anaerobic bacteria cells, in thefirst cell separator 420 is delivered via the outlet line 426 to therupturing device 460.

Inside the processing chamber 450, the cell-free permeate solution isprocessed into an oxygenated hydrocarbonaceous compound. The processingchamber 450 also recycles water back to the fermentation vessel 410 viathe outlet line 454. In total, the processing chamber 450 sends out 95%ethanol through the outlet line 452 for further downstream processing.

Inside the rupturing device 460, the cell membranes of the anaerobicbacterial cells contained within the cell-containing suspension areruptured to generate a homogenate. The homogenate is sent to thefractionator 470 through the outlet line 462. In one aspect, the outletline 474 is connected to the fractionator 470 that delivers a firstprotein-containing portion to be produced as the protein-rich nutrientsupplement. The outlet line 472 is connected to the fractionator 470that allows a cell debris portion to flow into another apparatus forfurther downstream processing. In another aspect, one or more ofprotein-containing fractions after one or more fractionators to removeunwanted contaminants and debris are added back together and produced asa protein-rich nutrient supplement.

Exemplary rupturing device that can be used herein includes, but is notlimited to, a microfluidics device, a sonication device, an ultrasonicdevice, a mechanical disruption device, a French press, a freezer, aheater, a pasteurization device, an UV sterilization device, a gamma raysterilization device, a reactor, a homogenizer, and combinationsthereof.

One example of the rupturing device 460 is a device that causes anirreversible change to the structure of cell membranes and cell walls ofbacterial microorganisms to allow further manipulation of the contentsof the bacterial cells. Contents of the bacterial cells include nucleicacids, proteins, glycogen, pigments, lipid droplets, crystals, and othernutrients, such as different forms of carbon, nitrogen, sulfur, calcium,etc.

In one aspect, the rupturing device 460 breaks open the cells byrupturing cell membranes of anaerobic bacterial cells by use of highforce. High shearing forces are applied to the anaerobic bacterial cellswithin the cell-containing suspension, such as by sound, pressure, ormechanical means. In the present invention, the method includes sendingto a rupturing device 460 a cell-containing suspension containing theanaerobic bacterial cells at a second concentration. The rupturingdevice 460 breaks open the cells by rupturing cell membranes of thecells with a strong force (e.g., mechanical, sound, pressure) andgenerating a homogenate, wherein there is better accessibility to usefulmoieties within the bacterial cells, e.g., protein, given the rupturedstate of the bacterial cells. Alternatively, the method includesdelivering the homogenate to a second rupturing device before thehomogenate is delivered to a first fractionator. The second rupturingdevice further ruptures the cells of the homogenate, after which aprotein-rich nutrient supplement is produced.

As an example, the rupturing device 460 is a microfluidics device. Themicrofluidics device includes, but is not limited to, reaction chambers,tubes, pumps, flanged pipes, rings, gaskets, high-pressure check valves.The reaction chamber of the microfluidics device can be a ceramicreaction chamber, an abrasion-resistant chamber, a spool reactionchamber, that is single-slotted, multi-slotted and has micro-channeling.

As another example, the rupturing device 460 is an enzymatic treatmentdevice. As yet another example, the rupturing device is an ultrasonicdevice. The ultrasonic device is an ultrasonic probe or an ultrasonicbath. The ultrasonic device shears cells by use of high frequency soundwaves to agitate and rupture cells. As yet another example, therupturing device is a freezing device. The freezing device has a freezeand thaw cycle, wherein the bacterial cells enter multiple rounds of thefreeze and thaw cycle, wherein the cells are frozen and then thawed in abuffer. As yet another example, the rupturing device is a mechanicalrupturing device. The mechanical rupturing device includes mechanicalblades or beads to break down cell walls and/or cell membranes of thebacterial cells.

Inside the fractionator 470, the homogenate is then fractionated into afirst protein-containing portion and a protein-containing cell debrisportion. Next, the first protein-containing cell debris portion isdelivered to the dehydration chamber 475A via the outlet line 472. Andthe first protein-containing portion is delivered to the dehydrationchamber 475B via the outlet line 474. Exemplary fractionators include,but are not limited to, various types of solid-liquid fractionators,centrifugation devices, continuous centrifuges, decanter centrifuges,disc-stack centrifuges, a filtration devices, a hollow fiber filtrationdevice, a spiral wound filtration device, a ceramic filter device, across-flow filtration device, a size exclusion device, one or series ofsize exclusion columns, one or series ion exchange columns, one orseries of carbon polymer columns, a flow-through magnetic fractionator,an ultrafiltration device, one or series of affinity chromatographycolumns, one or series of gel filtration columns, and combinationsthereof.

After the dehydration process undergone in the dehydration chamber 475Aand dehydration chamber 475B, a protein-rich nutrient supplement can beobtained and collected via both of the outlet lines 476A and 476B, eachfrom the dehydration chamber 475A and the dehydration chamber 475B,respectively.

FIG. 4B shows a schematic of a bacterial fermentation system 400B withone fermentation vessel, two cell separators, one processing chamber,one rupturing device, two fractionators, and three dehydration chambersto obtain protein-rich nutrient supplements from a bacterialfermentation process. The bacterial fermentation system 400B includesthe inlet line 402, the inlet line 404, the fermentation vessel 410, theoutlet line 412, the outlet line 414, an outlet line 416, a first cellseparator 420, the outlet line 422, the outlet line 424, the outlet line426, a second cell separator 430, an outlet line 432, an outlet line434, an outlet line 436, the processing chamber 450, the outlet line452, the outlet line 454, the rupturing device 460, the outlet line 462,a first fractionator 470, the outlet line 472, the outlet line 474, thedehydration chamber 475, the outlet line 476, a second fractionator 480,an outlet line 482, an outlet line 484, a dehydration chamber 485A, anoutlet line 486A, a dehydration chamber 485B, and an outlet line 486B.

In one aspect, the cell separator 430 is a cell concentrator. For thepresent invention, the method includes collecting from the fermentationvessel an amount of a fermentation liquid broth containing the cells ofthe anaerobic bacteria at a first concentration. This collection isdelivered through the outlet line 416 that connects the fermentationvessel 410 to the cell separator 430. In the cell separator 430, thefermentation liquid broth is separated into a cell-free permeatesolution and a cell-containing suspension containing the anaerobicbacterial cells at a first concentration and concentrated to a secondconcentration (for example, with a high concentration of cells, higherthan the first concentration of the fermentation liquid broth). Thecell-free permeate solution is sent to the processing chamber 450through the outlet line 432 that connects the processing chamber 450 andthe cell separator 430. The cell-containing suspension containing thecells at the second concentration is sent to the rupturing device 460through the outlet line 436 that connects the rupturing device 460 tothe second cell separator 430.

In one aspect, after being processed by the rupturing device 460, thehomogenate is delivered to the fractionator 470 to separate into aprotein-containing portion and a cell debris portion. The fractionator470 is connected to the rupturing device 460 via the outlet line 462.The fractionator 470 has at least two outlet lines, where the outletline 472 is used to deliver the cell debris portion and the secondoutlet line 474 is used to deliver the protein-containing portion.

In one aspect, the first protein-containing portion is delivered to asecond fractionator 480 to further separate out a secondprotein-containing portion from the first protein-containing portion.The second fractionator 480 is connected to the first fractionator 470via an outlet 474. The second fractionator 480 has at least two outlets,wherein from a first outlet 482 flows cell debris and from a secondoutlet 484 flows a second protein-containing portion. The method furtherincludes collecting the second protein-containing portion from thesecond fractionator. In still another aspect, there are two or morefractionators. In yet another aspect, there is only one fractionatorwithin the bacterial fermentation system used for the present invention,from which a first protein-containing portion is collected. Exemplaryfractionators include, but are not limited to, various types ofsolid-liquid fractionators, centrifugation devices, continuouscentrifuges, decanter centrifuges, disc-stack centrifuges, a filtrationdevices, a hollow fiber filtration device, a spiral wound filtrationdevice, a ceramic filter device, a cross-flow filtration device, a sizeexclusion device, one or series of size exclusion columns, one or seriesion exchange columns, one or series of carbon polymer columns, aflow-through magnetic fractionator, an ultrafiltration device, one orseries of affinity chromatography columns, one or series of gelfiltration columns, and combinations thereof.

FIG. 4C shows a schematic of a bacterial fermentation system 400C withone fermentation vessel, one cell separator, one cell-free holding tank,one processing chamber, one rupturing device, one fractionator, and twodehydration chambers to obtain protein-rich nutrient supplements from abacterial fermentation process. The bacterial fermentation system 400Cincludes the inlet line 402, the inlet line 404, the fermentation vessel410, the outlet line 412, the outlet line 414, the cell separator 420,the outlet line 422, the outlet line 424, the outlet line 426, acell-free holding tank 440, an outlet line 442, an outlet line 444, theprocessing chamber 450, the outlet line 452, the outlet line 454, therupturing device 460, the outlet line 462, the fractionator 470, theoutlet line 472, the outlet line 474, a dehydration chamber 475A, anoutlet line 476A, a dehydration chamber 475B, and an outlet line 476B.

FIG. 4D shows a schematic of a bacterial fermentation system 400D withone fermentation vessel, one cell separator, one cell-free holding tank,one processing chamber, one rupturing device, two fractionators, andthree dehydration chambers to obtain protein-rich nutrient supplementsfrom a bacterial fermentation process. The bacterial fermentation system400D includes the inlet line 402, the inlet line 404 the a fermentationvessel 410, the outlet line 412, the outlet line 414, the cell separator420, the outlet line 422, the outlet line 424, the outlet line 426, thecell-free holding tank 440, the outlet line 442, the outlet line 444,the processing chamber 450, the outlet line 452, the outlet line 454,the rupturing device 460, the outlet line 462, a first fractionator 470,the outlet line 472, the outlet line 474, the dehydration chamber 475,the outlet line 476, a second fractionator 480, the outlet line 482, theoutlet line 484, the dehydration chamber 485A, the outlet line 486A, thedehydration chamber 485B, and the outlet line 486B.

FIG. 4E shows a schematic of a bacterial fermentation system 400E withone fermentation vessel, two cell separators, one cell-free holdingtank, one processing chamber, one rupturing device, two fractionators,and three dehydration chambers to obtain protein-rich nutrientsupplements from a bacterial fermentation process. The bacterialfermentation system 400E includes the inlet line 402, the inlet line404, the fermentation vessel 410, the outlet line 412, the outlet line414, the outlet line 416, a first cell separator 420, the outlet line422, the outlet line 424, a second cell separator 430, the outlet line432, the outlet line 436, the cell-free holding tank 440, the outletline 442, the outlet line 444, the processing chamber 450, the outletline 452, the outlet line 454, the rupturing device 460, the outlet line462, a first fractionator 470, the outlet line 472, the outlet line 474,the dehydration chamber 475, the outlet line 476, a second fractionator480, the outlet line 482, the outlet line 484, the dehydration chamber485A, the outlet line 486A, the dehydration chamber 485B, and the outletline 486B.

FIG. 4F shows a schematic of a bacterial fermentation system 400F withone fermentation vessel, one cell separator, one cell-free holding tank,one processing chamber, one cell-containing holding tank, one rupturingdevice, two fractionators, and three dehydration chambers to obtainprotein-rich nutrient supplements from a bacterial fermentation process.The bacterial fermentation system 400F includes the inlet line 402, theinlet line 404, an inlet line 406, the fermentation vessel 410, theoutlet line 412 the outlet line 414, the cell separator 420, the outletline 422, the outlet line 424, the outlet line 426, the cell-freeholding tank 440, the outlet line 442, the outlet line 444, acell-containing holding tank 445, an outlet line 446, the processingchamber 450, the outlet line 452, the outlet line 454, the rupturingdevice 460, the outlet line 462, the fractionator 470 the outlet line472, the outlet line 474, the dehydration chamber 475, the outlet line476, the fractionator 480, the outlet line 482, the outlet line 484, thedehydration chamber 485A, the outlet line 486A, the dehydration chamber485B, and the outlet line 486B.

In one aspect, the bacterial fermentation system 400F includes a holdingtank (e.g., the cell-containing holding tank 445) to house bacterialcells sent from the bacterial fermentation vessel. In one embodiment,the holding tank is a storage vessel that stores anaerobic bacterialcells of the microbial biomass collected from the fermentation vessel.This alleviates concerns over bottleneck issues when the bacterial cellsare traveling from the fermentation vessel 410 into the rupturing device460, with the bacterial cells being continuously collected from thebacterial fermentation vessel 410 and delivered to the rupturing device460 without overloading the rupturing device 460. The delivery rate ofthe concentrated bacterial cells into the rupturing device 460 may be ata lower rate than the delivery rate of the fermentation liquid broth outof the cell separator/concentrator.

In another embodiment, the holding tank (e.g., the cell-containingholding tank 445) serves as a pretreatment device, where the bacterialcells are subjected to one or more additives to increase rupturingefficiency. Treating cells with additives makes the cell membrane moremalleable to mechanical disruption of the bacterial cells. Thispretreatment can take place before the cell-containing suspension entersinto the rupturing device 460. Alternatively, the pretreatment can takeplace after the cell-containing suspension is ruptured by the rupturingdevice 460 and a homogenate containing the anaerobic bacterial cells aretreated with one or more additives. Examples of the cell-containingholding tank 445) include, but are not limited to, process chambers,tanks, stainless steel tanks, plastic tanks, etc.

In the bacterial fermentation system 400F, the cell-containingsuspension containing the cells can pre-treated with one or moreadditives inside the cell-containing holding tank 445. The one or moreadditives may be added to the cell-containing holding tank 445 via theinlet line 406, for example, surfactants, detergents, EDTA, TritonX-100, Tween-20, sodium dodecyl sulfate, CHAPS, enzymes, proteases,lysozyme, benzonase, nuclease, ribonucleases (RNases),deoxyribonucleases (DNases), hydrolysis-inducing agents, pH-adjustingagents, and a combination thereof. One example of an additive as apH-adjusting agent is sodium hydroxide. Another example of an additiveas a pH-adjusting agent is hydrogen chloride.

In one aspect, the pretreatment device is connected to the cellseparator/concentrator (e.g., the cell separator 420 and/or the cellseparator 430), which is connected to the fermentation vessel 410. Inanother aspect, the pretreatment device is connected to the fermentationvessel 410 directly, wherein a component of the pretreatment device is acell separator and concentrator. The pretreatment device includes apretreatment chamber and inlets (e.g., the inlet line 406) to introducespecific additives to render the cell membranes of the bacterial cellsmore malleable to other rupturing techniques. The type of additive(s)used and the type of rupturing device used can be any number ofcombinations to increase the rupturing efficiency of the cells.

FIG. 4G shows a schematic of a bacterial fermentation system 400G withone fermentation vessel, two cell separators, one cell-free holdingtank, one processing chamber, one cell-containing holding tank, onerupturing device, two fractionators, and three dehydration chambers toobtain protein-rich nutrient supplements from a bacterial fermentationprocess. The bacterial fermentation system 400G includes the inlet line402, the inlet line 404, the inlet line 406, the fermentation vessel410, the outlet line 412, the outlet line 414, the outlet line 416, thecell separator 420, the outlet line 422, the cell separator 430, theoutlet line 432, the outlet line 436, the cell-free holding tank 440, anoutlet line 442, the outlet line 444, the cell-containing holding tank445, the outlet line 446, the processing chamber 450, the outlet line452, the outlet line 454, the rupturing device 460, the outlet line 462,the fractionator 470, the outlet line 472, the outlet line 474, thedehydration chamber 475, the outlet line 476, the fractionator 480, theoutlet line 482, the outlet line 484, the dehydration chamber 485A, theoutlet line 486A, the dehydration chamber 485B, and the outlet line486B.

FIG. 4H shows a schematic of a bacterial fermentation system 400H withone fermentation vessel, two cell separators, one cell-free holdingtank, one processing chamber, one cell-containing holding tank, onerupturing device, two fractionators, and three dehydration chambers toobtain protein-rich nutrient supplements from a bacterial fermentationprocess. The bacterial fermentation system 400H includes the inlet line402, the inlet line 404, the fermentation vessel 410, the outlet line412, the outlet line 414, the cell separator 420, the outlet line 422,the cell separator 430, the outlet line 432, the outlet line 436, thecell-free holding tank 440, the outlet line 442, the outlet line 444,the processing chamber 450, the outlet line 452, the outlet line 454,the rupturing device 460, the outlet line 462, an outlet line 464, thefractionator 470, the outlet line 472, the outlet line 474, thedehydration chamber 475, the outlet line 476, the fractionator 480, anoutlet line 482, the outlet line 484, the dehydration chamber 485A, theoutlet line 486A, the dehydration chamber 485B, and the outlet line486B.

In certain embodiments, the bacterial fermentation system 400H furtherincludes one or more recycle lines. In one embodiment, the recycle lineis a return line 464 connected to the rupturing device 460. The returnline 464 takes a portion of the product mixtures from the rupturingdevice and reenters into the rupturing device 460. This allows formultiple passes through the rupturing device 460, which, in turn,increases the ruptured amounts and protein concentrations of theprotein-containing homogenate of anaerobic bacterial cell and ensuresadequate accessibility to the protein compounds within the bacterialcells for further processing.

One or more rupturing devices can be used alone or in combination withone another. In other words, there can be one or more rupturing devicesin the bacterial fermentation system. In one respect, there are one ormore rupturing devices. In another respect, there is one rupturingdevice and a pretreatment device. In yet another respect, there are twoor more rupturing devices.

In certain embodiments, the bacterial fermentation system 400H furtherincludes one or more recycle lines. In one embodiment, the recycle lineis a return line 464 connected to the rupturing device 460. The returnline 464 takes a portion of the product mixtures from the rupturingdevice and reenters into the rupturing device 460. This allows formultiple passes through the rupturing device 460, which, in turn,increases the ruptured amounts and protein concentrations of theprotein-containing homogenate of anaerobic bacterial cell and ensuresadequate accessibility to the protein compounds within the bacterialcells for further processing.

FIG. 5A shows a schematic of a bacterial fermentation system 500A withone fermentation vessel, one cell separator, one processing chamber, onecell-containing holding tank, one rupturing device, one fractionator,and two dehydration chambers to obtain protein-rich nutrient supplementsfrom a bacterial fermentation process. The bacterial fermentation system500A includes an inlet line 502, an inlet line 504, a fermentationvessel 510, an outlet line 512, an outlet line 514, a cell separator520, an outlet line 522, an outlet line 524, an outlet line 526, aprocessing chamber 550, an outlet line 552, an outlet line 554, acell-containing holding tank 545, an inlet line 506, an outlet line 542,a mixer 548, a rupturing device 560, an outlet line 562, an outlet line564, a fractionator 570, an outlet line 572, an outlet line 574, adehydration chamber 575A, an outlet line 576A, a dehydration chamber575B, and an outlet line 576B.

In the cell separator 520, the fermentation liquid broth from thefermentation vessel 510 is separated into a cell-free permeate solutionand a cell-containing suspension containing the anaerobic bacterialcells at a second concentration. The cell-free permeate solution is sentto the processing chamber 550 through the outlet line 522 that connectsthe processing chamber 550 and the cell separator 520. Thecell-containing suspension containing the anaerobic bacterial cells at asecond concentration is sent to a processing chamber 550 through anoutlet 522 that connects the holding tank 550 to the cell separator 520.

The rupturing device 560 that delivers a homogenate is connected to afirst fractionator 570. In the first fractionator 570, the method 500Aincludes separating the cell-containing suspension into a firstprotein-containing portion and a cell debris portion. The firstfractionator 570 is connected to the rupturing device 560 via an outlet562. The first fractionator 570 has at least two outlets, wherein from afirst outlet 574 flows cell debris and from a second outlet 572 flows afirst protein-containing portion. Types of fractionators used include,but are not limited to, various types of solid-liquid fractionators,centrifugation devices, continuous centrifuges, decanter centrifuges,disc-stack centrifuges, a filtration devices, a hollow fiber filtrationdevice, a spiral wound filtration device, a ceramic filter device, across-flow filtration device, a size exclusion device, one or series ofsize exclusion columns, one or series ion exchange columns, one orseries of carbon polymer columns, a flow-through magnetic fractionator,an ultrafiltration device, one or series of affinity chromatographycolumns, one or series of gel filtration columns, and combinationsthereof.

In one example, the bacterial fermentation system 500A further includesthe cell-containing holding tank 545 serving as a pretreatment chamber.The cell-containing suspension containing anaerobic bacterial cells istreated with one or more additives in the cell-containing holding tank545. The cell-containing holding tank 545 is connected to the inlet line506 that supplies one or more additives (e.g., detergents, enzymes,buffers, pH-adjusting agents etc.). The inlet 506 is generally turnedoff and can be turned on when needed. The cell-containing holding tank545 holds the cell-containing suspension containing anaerobic bacterialcells until the cells reach high cell density (high concentrations) andcan serve to provide timed deliveries of specific amounts of thecell-containing suspension to the rupturing device 560.

The mixer 548 inside the cell-containing holding tank 545 is anagitating device, such as an agitating device with propeller inside. Therupturing device 560 is connected to the cell-containing holding tank545 via the outlet line 542. The rupturing device 560 generates ahomogenate of the anaerobic bacterial cells. After a specific durationwithin the cell-containing holding tank 545, the cell-containingsuspension is delivered to the rupturing device 560.

In one aspect, the fractionator 570 is connected to one or moredehydration chambers (e.g., the dehydration chambers 575A, 575B), whichreceive one or more protein-containing portions and dry them. Dryingtechniques used include drying, spray drying, lyophilizing, etc. Theprotein-containing portion is then further processed and blended intoprotein-rich nutrient supplements. The protein-containing portions mayhave a protein content of 10% or higher (such as between 10% and 80% orbetween 50% and 95%) of the protein-rich nutrient supplements.

FIG. 5B shows a schematic of a bacterial fermentation system 500B withone fermentation vessel, one cell separator, one processing chamber, onerupturing device, one cell-containing holding tank, one fractionator,and two dehydration chambers to obtain protein-rich nutrient supplementsfrom a bacterial fermentation process. The bacterial fermentation system500B includes the inlet line 502, the inlet line 504, the fermentationvessel 510, the outlet line 512, the outlet line 514, the cell separator520, the outlet line 522, the outlet line 524, the outlet line 526, theprocessing chamber 550, the outlet line 552, the outlet line 554, thecell-containing holding tank 545, the inlet line 506, the outlet line542, the mixer 548, the rupturing device 560, the outlet line 562, theoutlet line 564, a fractionator 580, an outlet line 582, an outlet line584, a dehydration chamber 585A, an outlet line 586A, an dehydrationchamber 585B, and an outlet line 586B. In this example, thecell-containing holding tank 545 is connected downstream of therupturing device 560 via the outlet line 562.

FIG. 5C shows a schematic of a bacterial fermentation system 500C withone fermentation vessel, one cell separator, one processing chamber, onecell-containing holding tank, one rupturing device, two fractionators,and three dehydration chambers to obtain protein-rich nutrientsupplements from a bacterial fermentation process. The bacterialfermentation system 500C includes an inlet line 502, an inlet line 504,a fermentation vessel 510, an outlet line 512, an outlet line 514, acell separator 520, an outlet line 522, an outlet line 524, an outletline 526, a processing chamber 550, an outlet line 552, an outlet line554, a cell-containing holding tank 545, an inlet line 506, a mixer 548,an outlet line 542, a rupturing device 560, an outlet line 562, anoutlet line 564, a first fractionator 570, the outlet line 572, theoutlet line 574, the dehydration chamber 575, the outlet line 576, asecond fractionator 590, an outlet line 592, an outlet line 594, adehydration chamber 595A, an outlet line 596A, a dehydration chamber595B, and an outlet line 596B.

FIG. 5D shows a schematic of a bacterial fermentation system 500D withone fermentation vessel, one cell separator, one processing chamber, onerupturing device, two fractionators, and four dehydration chambers toobtain protein-rich nutrient supplements from a bacterial fermentationprocess. The bacterial fermentation system 500D includes the inlet line502, the inlet line 504, the fermentation vessel 510, the outlet line512, the outlet line 514, the cell separator 520, the outlet line 522,the outlet line 524, the outlet line 526, the processing chamber 550,the outlet line 552, the outlet line 554, the rupturing device 560, theoutlet line 562, the outlet line 564, the fractionator 570, the outletline 572, the outlet line 574, a first fractionator 570, an outlet line592A, an outlet line 594A, a second fractionator 590, an outlet line592B, an outlet line 594B, a dehydration chamber 595A, an outlet line596A, a dehydration chamber 595B, an outlet line 596B, a dehydrationchamber 595C, an outlet line 596C, a dehydration chamber 595D, and anoutlet line 596D. In one aspect, the rupturing device 560 has a recyclestream line (e.g., the outlet line 564) that allows for multiple passesthrough the rupturing device 560.

FIG. 5E shows a schematic of a bacterial fermentation system 500E withone fermentation vessel, two cell separators, one processing chamber,one cell-containing holding tank, one rupturing device, twofractionators, and three dehydration chambers to obtain protein-richnutrient supplements from a bacterial fermentation process. Thebacterial fermentation system 500E includes the inlet line 502, theinlet line 504, the fermentation vessel 510, the outlet line 512, theoutlet line 514, an outlet line 516, the cell separator 520, the outletline 522, the outlet line 524, the processing chamber 550, the outletline 552, the outlet line 544, a cell separator 530, an outlet line 532,an outlet line 534, an outlet line 536, the cell-containing holding tank545, the inlet line 506, the outlet line 542, the mixer 548, therupturing device 560, the outlet line 562, the fractionator 570, theoutlet line 572, the outlet line 574, the dehydration chamber 575, theoutlet line 576, the fractionator 580, the outlet line 582, the outletline 584, the dehydration chamber 585A, the outlet line 586A, thedehydration chamber 585B, and the outlet line 586B.

FIG. 6 shows a schematic of a bacterial fermentation system 600 with onefermentation vessel, two cell separators, one cell-free holding tank,one processing chamber, one cell-containing holding tank, one rupturingdevice, two fractionators, and two dehydration chambers to obtainprotein-rich nutrient supplements from a bacterial fermentation process.The bacterial fermentation system 600 includes an inlet line 602, aninlet line 604, a fermentation vessel 610, an outlet line 612, an outletline 614, an outlet line 616, a first cell separator 620, an outlet line622, an outlet line 624, a cell-free holding tank 640, an outlet line642, an outlet line 644, a processing chamber 650, an outlet line 652,an outlet line 654, a second cell separator 630, an outlet line 632, anoutlet line 636, a cell-containing holding tank 645, an inlet line 606,an outlet line 646, a rupturing device 660, an outlet line 662, a firstfractionator 670, an outlet line 672, an outlet line 674, a dehydrationchamber 675, an outlet line 676, a second fractionator 690, an inletline 692, an outlet line 694, a dehydration chamber 695, and an outletline 696.

In one embodiment, there is one rupturing device that is a microfluidicsdevice, wherein the cells enter the rupturing device and are subjectedto high shearing forces in a reaction chamber to break apart the cellwalls and cell membranes of the anaerobic bacteria. The rupturedbacterial cells are then further processed via centrifugation,filtration, various methods of dehydrating the anaerobic bacterial cells(e.g., drying, freeze drying, lyophilizing, etc.), blending, the removalof heavy metal ions, incorporation as a nutrient supplement into aningestible substance, or combinations thereof.

In another embodiment, the bacterial fermentation system has two or morerupturing devices. The first rupturing device also can be a holding tankor a storage vessel that is holding the bacterial cells within acell-containing suspension that has been separated from the fermentationliquid. There is a first rupturing device that is a pretreatment device,wherein a cell-containing suspension enters the first rupturing deviceand is treated with additives to increase rupturing efficiency.Additives used include, but are not limited to, one or more detergents,enzymes, chemicals, or combinations thereof. There is a second rupturingdevice that is a microfluidics device, wherein the cells enter thesecond rupturing device and is subjected to high shearing forces in areaction chamber. The cell-containing suspension is forced throughmicro-channeling that causes the cell walls and cell membranes ofbacterial cells to rupture and break open, wherein contents of thebacterial cells become free-floating throughout the fermentation liquid.This permits collection of a first protein recovery, which can befurther manipulated by centrifugation, filtration, dehydration, etc.

III. Composition of Nutrient Supplements Comprising Fermentation-DerivedProteins

One or more protein-containing portions recovered from the bacterialfermentation system described herein may be subject to direct blendingwith a feedstock composition, drying, settling, filtration,ultrafiltration, microfiltration, vacuum filtration, centrifugation,sequential centrifugation, freeze drying, freezing, hydrolysis, andcombinations thereof to generate and obtain much pure forms of proteinsand at higher protein concentrations. In the aspect where the microbialbiomass is hydrolyzed, hydrolysis may be carried out via heat treatment,acid hydrolysis, enzyme hydrolysis, alkaline hydrolysis, andcombinations thereof.

In one embodiment of the method, the first protein-containing portion isproduced as the protein-rich nutrient supplement. The firstprotein-containing portion has a protein content that is between 60% to80%. In another aspect, the first protein-containing portion has aprotein content that is between 40% to 60%. In yet another aspect, thefirst protein-containing portion has a protein content that is between10% to 40%. In another embodiment the protein-containing portioncomprises a protein content of between about 10% to about 95% of thecomposition, a carbohydrate content of between about 5% to about 35% ofthe composition, and a nucleic acid content of less than 15% of thecomposition.

In one embodiment, the present invention provides a composition that isa protein-rich nutrient supplement. This composition is generated from afermentation process using acetogenic bacterial culture. Thiscomposition comprises a protein-containing portion separated from a celldebris portion of a homogenate, wherein the homogenate is obtained fromrupturing a cell-containing suspension containing cells of theacetogenic bacterial culture, and wherein the cell-containing suspensionis obtained from a fermentation liquid being delivered out of afermentation vessel during fermentation of a gaseous substrate using theacetogenic bacterial culture.

In one aspect, the acetogenic bacterial culture is selected from a groupconsisting of Clostridium bacteria, Acetobacterium bacteria, andcombinations thereof. The gaseous substrate fermented comprises one ormore gases selected from the group consisting of carbon sourcesubstrates, carbon monoxide (CO), carbon dioxide (CO₂), hydrogen (H₂)gas, syngas, and combinations thereof.

In another aspect, the protein-containing portion of the compositioncomprises a protein content of between about 10% to about 80% of thecomposition, a carbohydrate content of between about 5% to about 35% ofthe composition, and a nucleic acid content of between about 5% to about15% of the composition. The protein content in the protein-containingportion is greater than a carbohydrate content in the protein-containingportion. In yet another aspect, the nucleic acid content is no more than2% of the composition. This is a composition ingestible for humans andanimals alike.

In another embodiment, the composition of a protein-rich nutrientsupplement comprises a purified protein product separated from a firstamount from a protein-containing portion and a second amount from a celldebris portion of a homogenate, wherein the homogenate is obtained fromrupturing a cell-containing suspension containing cells of theacetogenic bacterial culture, and wherein the cell-containing suspensionis obtained from a fermentation liquid being delivered out of afermentation vessel during fermentation of a gaseous substrate using theacetogenic bacterial culture. The cell debris portion comprises cellwall particulates, cell membrane particulates, protein aggregates,inclusion bodies, nucleic acid, and other components of an anaerobicbacterial cell. The fermentation liquid broth delivered out of thefermentation vessel is separated into a cell-free permeate solution andthe cell-containing suspension containing the cells of the acetogenicbacterial culture. In one aspect, the partially purified protein producthas a nucleic acid content that is no more than 2%. In another aspect,the nucleic acid content is no more than 8% to 12%.

In one aspect, the composition includes a protein content of betweenabout 10% to about 80% of the composition, a carbohydrate content ofbetween about 5% to about 35% of the composition, and a nucleic acidcontent of between about 5% to about 15% of the composition. The proteincontent in the protein-containing portion is greater than a carbohydratecontent in the protein-containing portion.

In another aspect, the composition includes a protein content of betweenabout 10% to about 80% of the composition, a carbohydrate content ofbetween about 5% to about 35% of the composition, and a nucleic acidcontent of no more than 2% of the composition.

In yet another embodiment, the feedstock composition when removed fromthe bacterial fermentation vessel provides about 220 kcal or more per100 grams of acetogenic biomass and may include about 15 grams or morecarbohydrate per 100 grams of acetogenic biomass, on a dry weight basis.In this aspect, the feedstock has a weight ratio of carbohydrates toprotein of about 1.0 or less. In another aspect, the feedstock includesabout 18 mg or more calcium per 100 grams of acetogenic biomass, about150 mg or more iron per 100 grams of cell mass, about 25 mg or moresodium per 100 grams of acetogenic biomass, about 1200 mg or morepotassium per 100 grams of biomass, or a combination thereof, on a dryweight basis. The feedstock composition includes both essential andnonessential amino acids. The feedstock composition may also includenucleotides.

In one aspect, the feedstock composition provides a protein content ofabout 60 grams or more per 100 grams of acetogenic biomass, in anotheraspect, about 60 to about 90 grams per 100 grams of acetogenic biomass,in another aspect, about 65 to about 85 grams per 100 grams ofacetogenic biomass, and in another aspect, about 70 to about 80 gramsper 100 grams of acetogenic biomass, all on a dry weight basis.

In another aspect, the feedstock composition provides about 220 kcal ormore per 100 grams of dry acteogenic biomass, in another aspect, about220 kcal to about 400 kcal, in another aspect, about 250 kcal to about350 kcal, in another aspect, about 300 kcal to about 325 kcal, and inanother aspect, about 220 kcal to about 300 kcal.

In another aspect, the feedstock composition provides about 15 grams ormore carbohydrates per 100 grams of dry acetogenic biomass, in anotheraspect, about 15 grams to about 60 grams, in another aspect, about 20 toabout 40 grams, in another aspect, about 25 to about 35 grams, and inanother aspect, about 30 to about 35 grams. In this aspect, thefeedstock includes a weight ratio of carbohydrates to proteins of about1.0 or less, in another aspect, about 0.75 or less, in another aspect,0.5 or less, in another aspect, about 0.25 or less, and in anotheraspect, 0.1 or less. In one aspect, the feedstock has no detectablecarbohydrates and only includes protein. In another aspect, thecarbohydrate may include ethanol and/or water-soluble sugars.

The feedstock composition may also include fiber. Fiber may include aciddetergent fiber, neutral detergent fiber, digestible fiber, and/orindigestible fiber. The feedstock composition may also include starch.In yet another aspect, the feedstock composition includes calcium, iron,sodium and potassium in the following amounts (all expressed as mg per100 grams of acetogenic biomass on a dry weight basis): Calcium: about18 mg or more, in another aspect, about 20 mg or more, in anotheraspect, about 25 mg or more, and in another aspect, about 30 mg or more;Iron: about 150 mg or more, in another aspect, about 175 mg or more, inanother aspect, about 200 mg or more, and in another aspect, about 225mg or more; Sodium: about 25 mg or more, in another aspect, about 30 mgor more, in another aspect, about 35 mg or more, and in another aspect,about 40 mg or more; Potassium: about 1200 mg or more, in anotheraspect, about 1300 mg or more, in another aspect, about 1400 mg or more,and in another aspect, about 1500 mg or more.

In one aspect, the feedstock composition may include de minimis amountsof metals. In an alternative aspect, the feedstock may include levels ofcertain desirable metals. Examples of metals that may or may not bepresent in the feedstock include zinc, molybdenum, cadmium, scandium,titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper,tungsten and selenium.

In another aspect, the acetogenic biomass may include any one of thefollowing amino acids, either alone or in any combination (expressed asgrams per 100 grams acetogenic biomass on a dry weight basis): EssentialAmino Acids Content: Arginine: in one aspect, about 2.5 grams or more,in another aspect, about 3.0 grams or more, in another aspect, about 3.5grams or more, in another aspect, about 4.0 grams or more, in anotheraspect, about 4.5 grams or more, in another aspect, about 5.0 grams ormore, in another aspect, about 6.0 grams or more, and in another aspect,about 7.0 grams or more; Histidine: in one aspect, about 1.5 grams ormore, in another aspect, about 2.0 grams or more, in another aspect,about 2.5 grams or more, in another aspect, about 3.0 grams or more, inanother aspect, about 3.5 grams or more, in another aspect, about 4.0grams or more, in another aspect, about 5.0 grams or more, and inanother aspect, about 6.0 grams or more; Isoleucine: in one aspect,about 4.0 grams or more, in another aspect, about 4.5 grams or more, inanother aspect, about 5.0 grams or more, in another aspect, about 5.5grams or more, in another aspect, about 6.0 grams or more, in anotheraspect, about 7.0 grams or more, in another aspect, about 8.0 grams ormore, and in another aspect, about 9.0 grams or more; Leucine: in oneaspect, about 4.5 grams or more, in another aspect, about 5.0 grams ormore, in another aspect, about 5.5 grams or more, in another aspect,about 6.0 grams or more, in another aspect, about 6.5 grams or more, inanother aspect, about 7.0 grams or more, in another aspect, about 8.0grams or more, and in another aspect, about 9.0 grams or more; Lysine:in one aspect, about 6.0 grams or more, in another aspect, about 6.5grams or more, in another aspect, about 7.0 grams or more, in anotheraspect, about 7.5 grams or more, in another aspect, about 8.0 grams ormore, in another aspect, about 9.0 grams or more, in another aspect,about 10.0 grams or more, and in another aspect, about 12.0 grams ormore; Methionine: in one aspect, about 1.5 grams or more, in anotheraspect, about 2.0 grams or more, in another aspect, about 2.5 grams ormore, in another aspect, about 3.0 grams or more, in another aspect,about 3.5 grams or more, in another aspect, about 4.0 grams or more, inanother aspect, about 5.0 grams or more, and in another aspect, about6.0 grams or more; Phenylalanine: in one aspect, about 2.5 grams ormore, in another aspect, about 3.0 grams or more, in another aspect,about 3.5 grams or more, in another aspect, about 4.0 grams or more, inanother aspect, about 4.5 grams or more, in another aspect, about 5.0grams or more, in another aspect, about 5.5 grams or more, and inanother aspect, about 6.0 grams or more; Threonine: in one aspect, about3.0 grams or more, in another aspect, about 3.5 grams or more, inanother aspect, about 4.0 grams or more, in another aspect, about 4.5grams or more, in another aspect, about 5.0 grams or more, in anotheraspect, about 6.0 grams or more, in another aspect, about 7.0 grams ormore, and in another aspect, about 8.0 grams or more; Tryptophan: in oneaspect, about 0.4 grams or more, in another aspect, about 0.5 grams ormore, in another aspect, about 0.6 grams or more, in another aspect,about 0.7 grams or more, in another aspect, about 0.8 grams or more, inanother aspect, about 0.9 grams or more, in another aspect, about 1.0grams or more, and in another aspect, about 1.5 grams or more; Valine:in one aspect, about 4.0 grams or more, in another aspect, about 4.5grams or more, in another aspect, about 5.0 grams or more, in anotheraspect, about 5.5 grams or more, in another aspect, about 6.0 grams ormore, in another aspect, about 7.0 grams or more, in another aspect,about 8.0 grams or more, and in another aspect, about 9.0 grams or more.

Other Amino Acids Content: Alanine: in one aspect, about 5.0 grams ormore, in another aspect, about 5.5 grams or more, in another aspect,about 6.0 grams or more, in another aspect, about 7.0 grams or more, inanother aspect, about 8.0 grams or more, in another aspect, about 9.0grams or more, in another aspect, about 10.0 grams or more, and inanother aspect, about 11.0 grams or more; Aspartic Acid: in one aspect,about 7.0 grams or more, in another aspect, about 7.5 grams or more, inanother aspect, about 8.0 grams or more, in another aspect, about 9.0grams or more, in another aspect, about 10.0 grams or more, in anotheraspect, about 11.0 grams or more, in another aspect, about 12.0 grams ormore, and in another aspect, about 14.0 grams or more; Cysteine: in oneaspect, about 1.0 grams or more, in another aspect, about 1.5 grams ormore, in another aspect, about 2.0 grams or more, in another aspect,about 2.5 grams or more, in another aspect, about 3.0 grams or more, inanother aspect, about 3.5 grams or more, in another aspect, about 4.0grams or more, and in another aspect, about 5.0 grams or more; Glutamicacid: in one aspect, about 9.0 grams or more, in another aspect, about9.5 grams or more, in another aspect, about 10.0 grams or more, inanother aspect, about 12.0 grams or more, in another aspect, about 14.0grams or more, in another aspect, about 16.0 grams or more, in anotheraspect, about 18.0 grams or more, and in another aspect, about 20.0grams or more; Glycine: in one aspect, about 3.0 grams or more, inanother aspect, about 3.5 grams or more, in another aspect, about 4.0grams or more, in another aspect, about 4.5 grams or more, in anotheraspect, about 5.0 grams or more, in another aspect, about 5.5 grams ormore, in another aspect, about 6.0 grams or more, and in another aspect,about 7.0 grams or more; Methionine: in one aspect, about 1.5 grams ormore, in another aspect, about 2.0 grams or more, in another aspect,about 2.5 grams or more, in another aspect, about 3.0 grams or more, inanother aspect, about 3.5 grams or more, in another aspect, about 4.0grams or more, in another aspect, about 5.0 grams or more, and inanother aspect, about 6.0 grams or more; Proline: in one aspect, about2.0 grams or more, in another aspect, about 2.5 grams or more, inanother aspect, about 3.0 grams or more, in another aspect, about 3.5grams or more, in another aspect, about 4.0 grams or more, in anotheraspect, about 4.5 grams or more, in another aspect, about 6.0 grams ormore, and in another aspect, about 7.0 grams or more; Serine: in oneaspect, about 2.5 grams or more, in another aspect, about 3.0 grams ormore, in another aspect, about 3.5 grams or more, in another aspect,about 4.0 grams or more, in another aspect, about 4.5 grams or more, inanother aspect, about 5.0 grams or more, in another aspect, about 5.5grams or more, and in another aspect, about 6.0 grams or more; Tyrosine:in one aspect, about 2.5 grams or more, in another aspect, about 3.0grams or more, in another aspect, about 3.5 grams or more, in anotheraspect, about 4.0 grams or more, in another aspect, about 4.5 grams ormore, in another aspect, about 5.0 grams or more, in another aspect,about 5.5 grams or more, and in another aspect, about 6.0 grams or more.

In one embodiment, the feedstock composition may be utilized asfeedstock in animal feed. In yet another embodiment, the feedstockcomposition may be utilized as feedstock in aquaculture. In yet anotherembodiment, the feedstock composition may be further processed andutilized as a nutrient supplement ingestible by animals and humansalike.

In one aspect, the present composition provides an effective amount ofnutrients to a bacterial fermentation process. In this aspect, an“effective amount” describes use in promoting a healthy fermentationprocess which may include at least one of the following: production oftotal alcohol at a STY of about 1 g or more total alcohol/(L·day);providing a cell density of about 2.0 grams/liter or more; andmaintaining the culture in a steady state. The bacterial fermentationprocess may be the fermentation of a CO-containing gaseous substrate andmay be the same bacterial fermentation process from which the feedstockwas originally derived.

In another aspect, the present composition provides an effective amountof nutrition to an animal. An “effective amount” describes use inpromoting healthy growth in an animal is an amount sufficient to promoteat least one of the following: inhibition of bacterial load in theanimal; prevention or decrease the incidence of necrotic enteritis inpoultry; stimulation of the immune response in the animal; enhancementof the effectiveness of antibiotics and vaccines administered to theanimal in feed or otherwise; increased growth rate per amount of feedadministered; increased milk production; decreases in mortality rate;and the like. Several factors may be considered, include but not limitedto such factors as the animal's age, level of activity, hormone balance,and general health in determining the effective amount, which istailored to the animal, for example by beginning with a low dosage andtitrating the dosage to determine the effective amount.

Animals that can benefit from ingesting the present composition include,for example, poultry such as chickens, ducks, geese, turkeys, quail,game hens, and the like; beef and dairy cattle, pigs, goats, and thelike; domestic animals such as dogs and cats; aquatic animals such assalmon, salmonids, trout, tilapia, shrimp, lobster and the like; and,humans. Uses of the protein-rich nutrient supplement include fatteningcows, pigs, poultry, and fish. Other uses of the present compositioninclude serving as emulsifying aids to improve the nutritive value of amultitude of consumable goods, including baked goods, soups, prepackagedmeals, smart foods, and diet foods. Still other uses include paperprocessing, leather processing, and foam stabilization.

EXAMPLES Example 1: A Continuous Bacterial Fermentation Process

A synthesis or waste gas containing CO and/or CO₂/H₂ is continuouslyintroduced into a stirred tank bioreactor containing a strain ofClostridium ljungdahlii, along with a fermentation medium containingvitamins, trace metals and salts. A suitable fermentation medium used isreported in Table 1 below.

During method start-up using a culture inoculum of 10% or less thereactor is operated with a batch liquid phase, where the fermentationmedium is not fed continuously to the reactor. The liquid phase in thereactor thus consists of a batch of fermentation medium with a nominalconcentration of one or more limiting nutrients, e.g., calciumpantothenate, cobalt. Alternatively, a rich medium containing yeastextract, trypticase, or other complex nutrients can also be employed.

Ideally, the gas phase at start-up is CO₂-free and contains excess H₂.The gas rate and agitation rate are kept at low levels (less than 500rpm in a New Brunswick Scientific Bioflo® fermentation bioreactor) toyield CO and H₂ in slight excess, but at the same time, avoiding COsubstrate inhibition. In a one-liter laboratory New Brunswick ScientificBioflo® fermentation bioreactor, as an example, where the feed gascomposition is 63% H₂, 32% CO and 5% CH₄, the agitation rate to initiatestart-up is 400 rpm and the gas rate is 20 ml/min. To bring aboutethanol production during start-up, there is in excess both H₂ andliquid nutrients. Limitations placed on certain nutrients within thefermentation medium take place at a later time. Thus, excess liquidnutrients (e.g., calcium pantothenate, cobalt) are actually presentduring start-up to avoid unwanted culture acclimation to low nutrients.

TABLE 1 FERMENTATION MEDIUM: COMPONENTS & CONCENTRATIONS 1x EtOH ConcComponent Provided As (ppm) NH4+ NH₄Cl•(NH₄)₂HPO₄ 838 Fe FeCl₂•4H₂O 16.8Ni NiCl₂•6H₂O 0.198 Co CoCl₂•6H₂O 0.991 Se Na₂SeO₃ 0.0913 Zn ZnSO₄•7H₂O0.455 Mo Na₂MoO₂•2H₂O 0.238 Mn MnCl₂•2H₂O 0.167 B H₃BO₃ 1.05 CuCuCl₂•2H₂O 0.149 W Na₂WO₄•2H₂O 1.12 K KCl 78.6 Mg MgCl₂•6H₂O 59.8 NaNaCl 78.7^(a) Ca CaCl₂•2H₂O 54.5^(b) Cysteine HCl Cysteine HCl 250 PO4−2H₃PO₄•(NH₄)₂HPO₄ 816 Vitamins Vitamin cocktail^(c) Variable^(d) ^(a)Na+concentration is from NaCl only. It does not include Na+ from the othercomponents such as Na2WO4•2H2O, ^(b)Ca+2 concentration does not includecalcium from pantothenic acid or calcium salt. ^(c)Vitamins solutioncontains d-biotin, thiamine HCl, and d-pantothenic acid, calcium salt.^(d)Varies considerably from 0.3-0.5 ml at inoculation to as much as0.7-0.8 ml at high gas rates.

As bacterial fermentation proceeds over a period of several hourspost-inoculation, CO₂ is produced from the conversion of CO, and H₂ isconsumed along with the CO₂, which is a signal to nominally increase theagitation rate to avoid gas mass transfer limitation. In the NewBrunswick Scientific Bioflo® CSTR, the exit gas is 25% CO, 67% H₂, 2%CO₂, and 6% CH₄. If the agitation rate is increased too quickly, COsubstrate inhibition occurs, as evidenced by a decrease in methaneconcentration after an increase in agitation. Thus, the agitation ratemight typically be increased by 200 rpm in 24 hours.

The procedure of monitoring CO₂ production (or H₂ conversion) whilenominally increasing agitation rate occurs at a relatively rapid rateuntil the target agitation rate is reached. A typical target agitationrate in the New Brunswick Scientific Bioflo® fermentation bioreactor is900 rpm. During this time of increasing agitation rate in batch liquidculture, monitoring cell production takes precedence over instigatingproduct formation. Thus, cell concentrations of about 1.5 g/L areattained, while typical product concentrations are 10 g/L ethanol and 2g/L acetate from the batch culture.

Once the target agitation rate is reached, the system is allowed to growto maximum H₂ uptake. It is desirable to have very high H₂ exitconcentrations (typically >60%) to assure ethanol production whilelimiting acetic acid production. The liquid fermentation medium feed isthen turned on (for systems having batch inoculation from stock culture)to initiate continuous liquid feed and the gas feed rate is increasedtoward the target flow rate. In the laboratory New Brunswick ScientificBioflo® fermentation bioreactor the liquid feed rate is typically 0.5ml/min, while the gas flow rate is increased by 10 to 15% every 24 hourstoward a target rate of 125 ml/min.

As the gas flow rate is increased, cell production increases until thereactor is eventually limited on liquid phase nutrients (e.g., calciumpantothenate, cobalt) as evidenced by a small drop in H₂ conversion, atthe target productivity. In the New Brunswick Scientific Bioflo® CSTR,this is recognized by a 10% drop in H₂ conversion at a targetproductivity of 20 g/L·day.

The production method and bacterial fermentation reactor system are thenmaintained at a steady state producing 15 to 35 g/L ethanol and 0 to 5g/L acetate as products, with only occasional small adjustments inlimiting nutrients, liquid rates and gas rate. Typical steady stateconditions in the laboratory New Brunswick Scientific Bioflo®fermentation bioreactor without cell recycle, are a gas retention time(gas flow rate/reactor liquid volume) of 20 minutes, a liquid retentiontime (liquid flow rate/reactor liquid volume) of 30 hours and anagitation rate of 900 rpm, yielding CO conversions of 92% and H₂conversions of 60% with a pantothenate limitation.

In one embodiment, cell recycle is added to the reactor system at thistime along with an adjustment in gas rate (increase) and a firstnutrient concentration (decrease). With cell recycle in the NewBrunswick Scientific Bioflo® CSTR, the gas retention time is typically 8minutes, the liquid retention time is 12 hours, the cell retention timeis 40 hours and the agitation rate is 900 rpm. These conditionstypically yield a CO conversion of 92% and a H₂ conversion of 50% with apantothenate limitation. This method of continuous fermentation allowsfor the continuous production and maintenance of high ethanolconcentrations with low by-product acetate concentrations under stableoperating conditions to enhance use of subject bacterial on anindustrial scale for ethanol production.

Example 2: Purging of Bacterial Cells from a Fermentation Vessel toControl Fermentation Product Ratios

A gaseous substrate (30% CO, 15% H₂, 10% CO₂, 45% N₂) fermentation takesplace in a CSTR (pH=5.0, Temperature=38° C., Pressure=20 psig) utilizingC. ljungdahlii, strain C-01, with cell recycle (cell retention time=40hours and the liquid retention time=6 hours) and the culture is notlimited in growth by cobalt, calcium pantothenate, or any othernutrient. As the culture grows, a cell density is attained such that thespecific uptake (mmol CO per gram of dry cells per minute) is below 0.5and acetic acid is produced preferentially to ethanol. To prevent thisoccurrence, the cell purge rate is increased to prevent an increase incell density, such that the steady concentration of cells is kept lowenough to maintain a specific uptake higher than 0.5 mmol CO per gramdry cells per minute. In doing so, the cell retention time is reduced tobetween 6 and 25 hours. See Table 2 for the monitoring of cellconcentration during a bacterial fermentation process of a strain of C.ljungdahlii.

TABLE 2 Cell Concentrations of Different Fermentation Liquid Broth fromVarious Cell Purges at Different Time Intervals Net Produc- % Cell Etha-Ace- Ace- tivity Time Water Conc.^(a) CO H₂ nol tate tate (g/L · (hr)Recycle (g/L) (%) (%) (g/L) (g/L) (g/L) day) 75 25 2.1 95 68 12 4 4 12193 50 2.1 95 75 15 6 5 15 462 75 2.1 92 60 17 5 4 17 554 50 1.6 85 3017→13 5 3 12-16 669 75 2.6 92 75 13→19 5 3 12-18 943 100 3.0 92 70 23 63 23 1087 100 3.0 92 60 23 6 0 23 1232 100 2.7 92 60 23 6 −0 23 1375 1003.0 91 60 27 6 −1 27 1534 100 3.5 88 35 23 5 0 23 ^(a)Dry cell weightbasis

Example 3: Analysis of Cell Biomass of Acetogenic Bacterial Cells

Clostridium ljungdahlii C-01 was grown in a bioreactor with syngas. Asample of the fermentate from the bioreactor and concentrated dry massof the cells biomass was analyzed in accordance with the followingprocedures in Table 3.

TABLE 3 Procedures for analyzing fermentate samples AOAC 990.08:Calcium, Iron, Sodium Heavy metals: ICP-M, based on AOAC 993.14 AOAC994.10: cholesterol AOAC 996.06: crude fat AOAC 992.16/991.43: dietaryfibers AOAC 980.13: sugars AOAC 926.08: moisture AOAC 990.03/992.23:protein AOCS Ce 1j-07 and Ce fat 1h-05, AOAC 996.06: 21 CFR 101.9:caloric content by calculation

The results of the analysis of the concentrated dry mass of the cellsbiomass are shown in Table 4.

TABLE 4 Results of Dry Weight of Biomass Results of analysis were asfollows: Content per 100 grams acetogenic biomass Analyte (dry weightbasis) carbohydrates 33.0 g calories (bomb calorimetry) 224.2 kcalprotein 60.4 g Calcium 18 mg Iron 152 mg Sodium 25 mg Potassium 1200 mg

The results of the amino acid analysis of the concentrated dry mass ofthe cells biomass are shown in Table 5.

TABLE 5 Results of Amino Acid Analysis of the Dry Weight of BiomassGrams per 100 grams acetogenic biomass (dry weight basis) Total aminoacids Aspartic acid 7.36 Threonine 3.29 Serine 2.83 Glutamic acid 9.18Proline 2.29 Glycine 3.28 Alanine 5.44 Valine 4.39 Methionine 1.83Isoleucine 4.44 Leucine 4.95 Tyrosine 2.58 Phenylalanine 2.90 Histidine1.70 Lysine 6.28 Arginine 2.77 Cysteine 1.05 Methionine 1.88 Tryptophan0.48 Free Amino Acids Glytamic acid 0.07

Example 4: Analysis of Protein, Carbohydrates, and Nucleic Acid Contentof Acetogenic Bacterial Cells

Clostridium ljungdahlii C-01 was grown in a bioreactor with syngas. Cellculture was centrifuged at 4,000 RPM to remove culture medium. Pelletswere collected and allowed to dry in an oven at 100 C overnight.

100 grams of crushed, dried pellet was sent for analysis using the sametests for carbohydrates and protein as described in Example 3. Table 6indicates that up to 80% of cell mass is protein.

TABLE 6 Three Components of Consideration in a Protein-Rich NutrientSupplement Test Number Carbohydrate Protein Nucleic Acid^(a) 1   33%≥60.6% ≥3% 2 7.06% ≥78.1% ≥3% 3 — ≥78.9% ≥3% ^(a)Rationale owing tothere being a non-protein nitrogen content that is less than 1%. Theratio of nitrogen in nucleic acid is about 3 g RNA/DNA to 1 g Nitrogen,such that the samples contained no more than 3% nucleic acid.

Example 5: Rupturing of Bacterial Cells by One or More MicroFluidization Rupturing Devices and the Protein Recovery for Rupturing ofBacterial Cells by the Micro Fluidization Rupturing Devices

The Microfluidizer by Microfluidics was identified as a rupturing deviceto rupture anaerobic bacterial cells from the fermentation process andproduce a protein-containing portion. A volume of fermentation liquidwas obtained from a fermentation vessel. Samples were concentrated1.5-fold by centrifugation or to a cell concentration of for example,about 20 g/L or larger. For example, about 15 g/L of a fermentationliquid was obtained from a fermentation vessel and Samples wereconcentrated by centrifugation to obtain a cell density of 22.4 g/L orhigher.

The resulting cells were re-suspended in solutions (e.g., into a 2 Lsolution which may contain about 44 g of cells) and sent toMicrofluidics. The microfluidization process involves rupturing cellswith high shear forces created by forcing the cells throughmicro-channels within the Microfluidics reaction chamber at highpressures.

Each sample was run at a different amount of time(s) and at a differentpressure. The pressures tested ranged between 10,000 and 30,000 poundsper square in (psi) for one or multiple passes. Each pass constitutes arun through the Microfluidizer. Pressure was supplied at a constant ratevia the rupturing device. The Microfluidizer generated six homogenizedsamples. The cell-containing suspensions can be treated with one or moreadditives (e.g., detergents, enzymes, etc.), and passed through theMicrofluidizer (e.g., at high shearing or pressures at 3,000 psi orlarger).

Several experiments were performed. Each sample was run at a differentamount of time(s) and at a different pressure. Among them, theconditions for six exemplary sample treatment experiments are shown: (1)a single pass at 18,000 psi; (2) two passes at 18,000 psi; (3) a singlepass at 23,000 psi; (4) two passes at 23,000 psi; (5) a single pass at28,000 psi; (6) two passes at 28,000 psi. Each pass constitutes anexperiment passing through the Microfluidizer. Pressure was supplied ata constant rate via the rupturing device. The resulting six homogenizedsamples of protein-containing fractions were generated after treatmentwith the Microfluidizer. Each sample of the protein-containing fractionwas analyzed for protein content using a Bradford assay.

In one experiment, some samples were treated with only one pass throughthe Microfluidizer. After one pass, a first protein-containing portionwas separated out of the homogenate. Then, the first protein-containingportion can be spray dried to obtain protein containing powder.

In a second experiment, samples were treated with two passes through theMicrofluidizer, where a pretreated cell-containing suspension flowedthrough a recycle stream to re-enter the Microfluidizer a second time.After two passes, a protein-containing portion was obtained. Then,powder form of the protein-containing portion can be obtained. Forexample, three different drying techniques (drying at high temperatures,spray drying, and lyophilizing) were tested after the protein-containingportion was obtained.

Example 6: Fractionating of the Homogenates of the Ruptured BacterialCells by One or More Filtration-Type Fractionator Devices

The homogenate of the protein-containing portion after the treatmentprocess of Example 5 was filtered through a nylon filter. Filtration ofthe homogenate allowed 5-15% of the original microbial biomass to berecovered as soluble protein, as indicated in Table 7.

TABLE 7 Percentage recovery of soluble proteins after filtration.Percent recovery of soluble proteins Sample Filtered 2,000 RPM 5,000 RPM10,000 RPM Run 1 5%-15% 10%-25% 5%-15% 5%-15% Run 2 5%-15% 10%-25%5%-15% 5%-15%

Example 7: Fractionating of the Homogenates of the Ruptured BacterialCells by One or More Centrifugation-Type Fractionator Devices

The homogenate of Example 4 underwent centrifugation at speeds between2,000 and 10,000 RPM for 6 minutes and the protein content was analyzedusing the Bradford protein assay. The percent of soluble proteinrecovery was calculated using the starting biomass concentration as abasis.

Results indicate that up to 25% of protein was recovered aftermicrofluidization, indicating that microfluidization followed bycentrifugation is a viable method to lyse cells and recover solubleprotein, also shown in Table 7.

As another example, a cell suspension with a cell density of 22.4 g/L(cells in fermentation broth) was sent to Microfluidics. One sample waspassed through a Microfluidizer at 18,000 psi and another sample waspassed through the same type of Microfluidizer twice at a pressure of28,000 psi. The results were compared and obtained as shown in Table 8.

For the samples passing one time at 18,000 psi, the proteinconcentration in the homogenate was 9.5 mg/mL. The sample was thenfiltered OR centrifuged and further analyzed. After filtration through a0.45 micron filter, the protein-rich fraction contained 2.4 mg/mL ofprotein. After centrifugation for 8 minutes at 2,000 RPM, the proteinconcentration of the protein-rich fraction is about 3.2 mg/mL protein.Higher centrifuge speeds resulted in less protein (at 5,000 RPM, theprotein concentration of the recovered fraction was about 1.0 mg/mL orhigher, or about 1.9 mg/mL or higher. At much higher centrifuge speed of10,000 RPM, the resulting protein concentration of the supernatantfraction after the spin was about 1.4 mg/mL).

TABLE 8 Protein Concentration (μg/mL) and Percentage (%) withinHomogenates (Mixtures of Ruptured Bacterial Cells) Filtered^(b) (0.45micron Centrifuged (rpm) Homogenate^(b) filter) 2,000 rpm 5,000 rpm10,000 rpm 18k, 1 pass 9502 μg/mL 2371 μg/mL 3178.4 μg/mL 1878.6 μg/mL1423.3 μg/mL Protein Recovery^(a) 42.4% 10.6% 14.2%  8.4% 6.35% 28k, 2passes 8549 μg/mL 1777 μg/mL 3006.9 μg/mL 2007.8 μg/mL 1330.3 μg/mLProtein Recovery^(a) 38.2% 7.9% 13.4% 8.96% 5.94% ^(a)Percentagescalculated by determining protein content of total cell mass.^(b)Homogenate and filtered samples were diluted by 11X prior tomeasuring.

Another group of samples were treated with two passes through theMicrofluidizer at 28,000 psi, where a previously lysed cell-containingsuspension flowed through a recycle stream to re-enter theMicrofluidizer a second time. The resulting protein concentration of theprotein-containing portion in the homogenate was measure at around 8.5mg/ml. The samples were then filtered and/or centrifuged. Afterfiltration through a 0.45 micron filter, the resulting proteinconcentration was about 1.8 mg/ml. After centrifugation for 8 minutes at2000 RPM, the resulting protein concentration was about 3.0 mg/ml.Higher centrifuge speed resulted in less protein (at 5000 RPM, it was2.0 mg/ml; at 10000 RPM it was 1.3 mg/ml).

Example 8: Cell Lysis from the Rupturing of Bacterial Cells by One orMore Micro Fluidization Rupturing Devices

The rupturing devices selected to conduct the rupturing process can bemicrofluidizer and other commercially available devices. In FIGS. 7B-7E,the rupturing devices selected to conduct the rupturing process is amicrofluidizer. The rupturing process inside the rupturing device can beconducted under different variables, including pressure and times ofpasses.

FIG. 7A illustrates an electron micrograph of the cell containingsuspension that was not subjected to lysis. The electronic micrographwas taken by a microscope and at an 100× magnification targeting thehomogenate.

The cell-containing suspension selected for undergoing the rupturingprocess inside the rupturing device can be of different densities. InFIG. 7A, the density of cell-containing suspension that did not undergoa rupturing process is about 10 g/L or higher, such as about 16 g/L orhigher.

FIG. 7B illustrates another electron micrograph of the homogenate whichis obtained inside the rupturing device 460, 560, or 660, and is aproduct from rupturing cell membranes of the anaerobic bacterial cellswithin the cell-containing suspension inside the rupturing device 460,560, or 660, according to one or more embodiments of the invention.

The electronic micrograph was taken by a microscope and at an 100×magnification targeting the homogenate. In this FIG. 7B, the rupturingdevices selected to conduct the rupturing process is a microfluidizer.In this FIG. 7B, the rupturing process is conducted at a pressure of1,000 psi or higher (e.g., between 1,000 pounds per square inch (psi) to9,000 psi or higher) against the cell-containing suspension and with onepass. Also, in FIG. 7B, the cell density of cell-containing suspensionselected for this rupturing process inside the rupturing device is about10 g/L or higher, such as at about 16 g/L or higher. Visually, there issignificant damage done to the cell membranes as a result of themicrofluidization process.

FIG. 7C illustrates an electron micrograph of the cell membranes of theanaerobic bacterial cells within the cell-containing suspension beforeit is ruptured inside the rupturing devices 460, 560, or 660, accordingto one or more embodiments of the invention.

The electronic micrograph was taken by a microscope and at an 100×magnification targeting the homogenate. In this FIG. 7C, the celldensity of the cell-containing suspension selected for this rupturingprocess inside the rupturing device is about 10 g/L or higher, such asat about 16 g/L or higher. The rupturing process is conducted at apressure of about 1,000 psi or higher (e.g., between 1,000 pounds persquare inch (psi) to 20,000 psi or higher) against the cell-containingsuspension and with one pass. Compared to the results in FIG. 7A, theresults as shown in FIG. 7C show significant rupturing of the cellmembranes occurred at this processing pressure.

FIG. 7D illustrates another electron micrograph of the homogenate whichis obtained inside the rupturing device 460, 560, or 660, and is aproduct from rupturing cell membranes of the anaerobic bacterial cellswithin the cell-containing suspension inside the rupturing device 460,560, or 660, according to one or more embodiments of the invention.

The electronic micrograph was taken by a microscope and at an 800×magnification targeting the homogenate. In this FIG. 7D, the rupturingdevices selected to conduct the rupturing process is a microfluidizer.In this FIG. 7D, the rupturing process is conducted at a pressure of1,000 psi (e.g., between 1,000 pounds per square inch (psi) to 22,000psi or higher) against the cell-containing suspension and with one pass.Also, in this FIG. 7D, the density of cell-containing suspensionselected for this rupturing process inside the rupturing device is about15 g/L or higher, such as at about 20 g/L or higher, or about 22.4 g/Lor higher.

FIG. 7E illustrates another electron micrograph of the homogenate whichis obtained inside the rupturing device 460, 560, or 660, and is aproduct from rupturing cell membranes of the anaerobic bacterial cellswithin the cell-containing suspension inside the rupturing device 460,560, or 660, according to one or more embodiments of the invention.

The electronic micrograph was taken by a microscope and at an 800×magnification targeting the homogenate. In this FIG. 7E, the rupturingdevices selected to conduct the rupturing process is a microfluidizer.In this FIG. 7E, the rupturing process is conducted at a pressure of thepressure of 1,000 psi (e.g., between 25,000 pounds per square inch (psi)to 30,000 psi or higher) against the cell-containing suspension and withtwo passes. Also, in this FIG. 7E, the cell density of cell-containingsuspension selected for this rupturing process inside the rupturingdevice is about 15 g/L or higher, such as at about 20 g/L or higher, orabout 23 g/L or higher.

Example 9: Rupturing of Bacterial Cells by One or More MicroFluidization Rupturing Devices with Pretreatment of the Bacterial Cellsin a Cell-Containing Holding Tank

The Microfluidizer rupturing device (e.g., Microfluidics device) wasused to rupture anaerobic bacterial cells from the fermentation processand produce a protein-containing portion. The fermentation liquid broth(e.g., at 15 g/L of cell density) was obtained from the high celldensity lab reactor of a fermentation vessel. Samples were concentratedby centrifugation to obtain a cell density of 45 g/L. The resulting cellsuspension (2 L containing 90 g of cells) was sent to a cell-containingholding tank for pretreatment.

The microfluidization process involves rupturing cells with high shearforces created by forcing the cells through a 1 micron reaction chamberat high pressures. Cell-containing suspensions containing the bacterialcells were split into six samples. The cell-containing suspensions weretreated with one or more additives (e.g., detergents, enzymes, etc.),and passed through the Microfluidizer at 15,000 psi. Only one passthrough the microfluidizer rupturing device was conducted. After onepass, a first protein-containing portion was separated out of thehomogenate and spray dried.

Example 10: Rupturing of Bacterial Cells by One or More SonicationRupturing Devices

A sonicator was identified as a rupturing device to rupture anaerobicbacterial cells from the fermentation process and produce aprotein-containing portion. 15 g/L fermentation liquid was obtained fromthe high cell density lab reactor of a fermentation vessel. Samples wereconcentrated by centrifugation to obtain a cell density of 22.4 g/L. Theresulting cell re-suspension (2 L containing 44 g of cells) wassubjected to sonication. The sonication process involves rupturing cellswith high force via sound energy at ultrasonic frequencies that agitatethe cells and break open the cell membranes. The cell-containingsuspension containing the bacterial cells was split into six samples.Each sample was subjected to sonication.

Example 11: Rupturing of Bacterial Cells by One or More SonicationRupturing Devices with Pretreatment of the Bacterial Cells in aCell-Containing Holding Tank

A sonicator was identified as a rupturing device to rupture anaerobicbacterial cells from the fermentation process and produce aprotein-containing portion. Fermentation liquid broth was obtained fromthree high cell density fermentation vessels. Samples were concentratedvia centrifugation to obtain cell densities between 4 and 10 mg/ml.

For example, a fermentation liquid broth obtained from a fermentationvessel (in this example, an exemplary vessel A) containing about 4.2mg/ml of bacterial cells was subjected to sonication in a variety ofbuffers. A cell-containing suspension from another fermentation vessel(an exemplary vessel B) of containing about 9.2 mg/ml of anaerobicbacterial cells was subjected to sonication in a variety of buffers. Acell-containing suspension from another fermentation vessel (anexemplary vessel C) of containing about 7.1 mg/ml of cells was alsosubjected to sonication in a variety of buffers.

After collection of fermentation broths from the fermentation vessel,the bacterial cells from the fermentation broth were spun down viacentrifugation (e.g., spinning down the bacterial cells at 4,000 RPM orhigher centrifugation speed) and re-suspended in their respectivebuffers.

Cells were resuspended in detergent-containing buffer (TrisHCl pH 8containing sodium dodecyl sulfate (SDS), CHAPS, Triton X-100, or Tween20) or enzyme-containing buffer (TrisHCl pH 8 containing lysozyme).TrisHCl pH 8 was used as the control buffer. The resulting cellsuspension was subjected to sonication.

Cells were sonicated in 5-second pulses followed by resting on ice inbetween. The cycle was repeated three times. After sonication, cellswere spun down for 10 minutes at 20K RPM and the supernatant wasremoved. The soluble protein fraction was analyzed for protein contentusing a Lowry-based protein assay. Percentages of soluble proteinrecovery were calculated based on concentration of cells subjected tosonication.

Several samples were also subjected to a freeze/thaw cycle, wherein thecells were completely frozen in Tris HCl buffer. After freezing at −80degrees Celsius, cells were completely thawed before re-freezing. Thiscycle was completed 5 times. After completion, cell-containingsuspension was spun down at 20,000 RPM for 10 minutes and thesupernatant was removed. Table 9 shows the protein recovery amounts bythe buffer type that the cell-containing suspension samples weresubjected to. Percentages of soluble protein recovery was calculatedbased on initial starting materials. Starting materials includefermentation liquid comprising a liquid nutrient medium, other essentialminerals, and an accumulation of acetogenic biomass.

Fermentation liquid was obtained from three high cell densityfermentation vessels. Samples were concentrated via centrifugation at4,000 RPM to obtain cell densities between 4 and 10 mg/ml. Cells wereresuspended in detergent-containing buffer (TrisHCl pH 8 containingsodium dodecyl sulfate (SDS), CHAPS, Triton X-100, or Tween 20) orenzyme-containing buffer (TrisHCl pH 8 containing lysozyme). TrisHCl pH8 was used as the control buffer. The resulting cell suspension wassubjected to sonication. The sonication process involves rupturing cellswith high force via sound energy at ultrasonic frequencies that agitatethe cells and break open the cell membranes.

Cells were sonicated in 5-second pulses followed by resting on ice inbetween. The cycle was repeated three times. After sonication, cellswere spun down for 10 minutes at 20,000 RPM and the supernatant wasremoved. The soluble protein fraction was analyzed for protein contentusing a Lowry-based protein assay. Percentages of soluble proteinrecovery were calculated based on concentration of cells subjected tosonication.

The results, shown in Table 9, indicate that detergent additives canincrease the solubility of proteins when cells are subjected tosonication. Specifically, the addition of SDS or lysozyme greatlyenhances the solubility of membrane proteins.

TABLE 9 Ruptured Protein Recovered by Percentage (%) Buffer Type VesselType 1 Vessel Type 2 Vessel Type 3 0.1-1% SDS 20-35 13-26  25-35  0.5-2%CHAPS  7-18 7-12 5-11 0.1-2% Triton X-100 10-25 5-10 5-13 2.5-5% Tween20 10-20 10-20  10-20  Control 15-20 8-13 8-13 Tris HCl  5-10 5-10 5-10Freeze/Thaw 25-30 15-20  15-20  Lysozyme

Example 12: Fractionating of the Homogenates of the Ruptured BacterialCells by One or More Filtration-Type Fractionator Devices

The homogenate of Example 4 is filtered through a nylon filter.Filtration of the homogenate allowed 10.7% of the original microbialbiomass to be recovered as soluble protein.

Example 13: Fractionating of the Homogenates of the Ruptured BacterialCells by One or More Centrifugation-Type Fractionator Devices

The homogenate of Example 4 underwent centrifugation at 2000 rpm, whichfractionated 3.2 mg of protein into supernatant. Centrifugation of thehomogenate allowed a 14.3% recovery of soluble protein separated out ofthe total cell mass of the initial microbial biomass collected from thefermentation vessel.

Example 14: Determination of pH Effect in the Cell-Containing Suspensionon Protein Recovered after Rupturing Through One or More RupturingDevices

Samples were collected from a high cell density fermentation vessel. Thecell concentration at the time of collection was approximately 22 g/L. Avolume of cell culture was spun down at 4,000 RPM for 10 minutes. Cellpellets were re-suspended in the same volume of TrisHCl as the volume ofculture media that was removed after centrifugation (so that sampleswere not concentrated or diluted). Multiple volumes of cell culture weresubjected to pH modification through addition of 0.5M NaOH so that thefinal pH of the culture broth was between 3.5 and 10. Samples wereprocessed between 10,000 and 20,000 psi for one or multiple passes. Eachsample was spun down at 13,300 RPM for 6 minutes. The supernatant wascollected and a Lowry-based protein assay was used to determine proteinconcentration in the soluble fraction.

Data, shown in Table 10, indicates that an increase in pH of cellhomogenate prior to microfluidization enhances protein solubility.Specifically, when the pH is high (above 7.6), the recovery of solubleprotein is enhanced.

TABLE 10 Ruptured Protein Recovered by Percentage (%) Sample Passesmg/ml Recovery % pH 3.5-5 1-4 2.5-3.5 12-15 pH 5-7.5 1-4  7-10 40-45 pH7.6-10 1-4 10-15 50-55 TrisHCl pH 8 1-4  9-15 40-60

Example 15. Determination of the Effect of Lysozyme on Soluble ProteinRecovery

Samples were collected from a high cell density fermentation vessel. Avolume of cell culture was spun down and re-suspended in TrisHCl bufferin the same manner as stated before. The pH of the culture broth wasincreased to pH 8 using 0.5M sodium hydroxide. To determine the effectof enzyme pre-treatment, 0.5 mg/ml lysozyme was used. For the culturebroth samples, incubation with lysozyme lasted ˜30 minutes at roomtemperature. For the TrisHCl samples, incubation with lysozyme lasted˜45 minutes at room temperature (difference due to delay in processingwith the microfluidizer as the bottleneck). Samples were processedbetween 10,000 and 20,000 psi for one or multiple passes. Controls werealso run where samples were not processed through the microfluidizer.Each sample was spun down at 13,300 RPM for 6 minutes. The supernatantwas collected and Lowry-based and Bradford-based protein assays wereused to determine protein concentration in the soluble fraction and theresults are shown in Table 11.

TABLE 11 Ruptured Protein Recovered by Percentage (%) Sample # of passesmg/ml % Protein Recovery Culture Broth 1-4 4-9 25-46 Culture Brothw/lysozyme 1-4 4.5-10  25-52 TrisHCl 1-4 4.5-9   26-46 TrisHClw/lysozyme 1-4  5-12 28-64 Reactor supernatant 0 0.5-1   0.02-4.5 TrisHCl w/lysozyme 0 2-5  4-25 Culture Broth 0 1-2 0.25-8.5  CultureBroth w/lysozyme 0 2-5  3-22

Example 16. Determination of Effect of Decreasing Lysozyme Concentrationand Extending Incubation Time

Samples were collected from a high cell density fermentation vessel andwere concentrated via centrifugation for 10 minutes at 4,000 RPM.Culture broth pH was adjusted to 7-10. Some samples were incubated at 37C with 100 ng/ml of lysozyme for an hour. Samples were taken every 10minutes to monitor lysozyme activity.

Large samples at 30 minutes and 1 hour were taken and processed throughthe microfluidizer at 10,000 to 20,000 psi for one or multiple passes.Each sample was spun down at 13,300 RPM for 6 minutes. The supernatantwas collected and a Lowry-based protein assay was used to determineprotein concentration in the soluble fraction and the results are shownin Table 12.

TABLE 12 Ruptured Protein Recovered by Percentage (%) Cell SolubleConcentration, Microfluidizer Protein Enzyme Sample g/L pressure, psimg/ml Recovery % None Supernatant — 0 0.5-1   — None Culture broth 10-150 1-2  7-10 10,000-20,000  5-10 42-52 40-50 0 1.5-3   3.5-4.510,000-20,000 15-20 35-46 Lysozyme Culture Broth 40-50 0 1.5-2.3 3.5-5  10,000-20,000 15-20 35-46

Example 17. Effect of pH and Cell Concentration on Extraction of Protein

Samples from the high cell density fermentation vessel were diluted toconcentrations between 1 and 10 g/L via centrifugation and resuspensionin culture media. The culture broth pH was adjusted to between 6 and 10using 0.5M sodium hydroxide. Samples (including un-modified culturebroth at fermentation pH) were processed between 10,000 and 20,000 psithrough the microfluidizer for one or multiple passes. Similarly,undiluted culture purge from the high cell density fermentation vesselwas processed at one acidic pH and one alkaline pH. Each sample was spundown at 13,300 RPM for 6 minutes.

The supernatant was collected and a Lowry-based protein assay was usedto determine protein concentration in the soluble fraction. Table 13indicates that as pH of the culture broth increases prior tomicrofluidization, the amount of protein recovered in the solublefraction increases.

FIG. 8A illustrates a graph of soluble protein obtained from therupturing device 460, 560 or 660 and from cell membranes of theanaerobic bacterial cells within the cell-containing suspension,according to one or more embodiments of the invention.

TABLE 13 Recovery of Ruptured Proteins (Protein Concentration indicated)pH of the Harvested Cells culture Ruptured Sample (Concentration: g/L)broth Protein (mg/ml) 1 1 4-5 0.92 2 1 6-7 1.03 3 1 8-9 1.23 4 5 4-5 1.35 5 6-7 2.7 6 5 8-9 3 7 10 4-5 1.7 8 10 6-7 4.3 9 10 8-9 5.0 10 18.5 4-57.1 10 18.5 8-9 8.9

The rupturing devices selected to conduct the rupturing process can bemicrofluidizer and other commercially available devices. In this FIG.8A, the rupturing devices selected to conduct the rupturing process is amicrofluidizer.

The rupturing process inside the rupturing device 460, 560 or 660 can beconducted under different variables, including pressure and times ofpasses. In this FIG. 8A, the rupturing process is conducted to thepressure of 15,000 pounds per square inch (psi) against thecell-containing suspension and under only one pass.

The cell-containing suspension selected for undergoing the rupturingprocess inside the rupturing device 460, 560 or 660 can be of differentdensities. In this FIG. 8A, four streams of cell-containing suspensionsare selected for this rupturing process inside the rupturing device 460,560 or 660. Each of the densities of the cell-containing suspensions are1 g/L, 5 g/L, 10 g/L and 18.5 g/L.

The y axis of the line 802 represents the yield of the protein from thecell homogenate ruptured and obtained from the rupturing device 460, 560or 660 prepared under different pH conditions (X-axis) from anaerobicbacterial cells within the cell-containing suspension with a density of18.5 g/L, according to one or more embodiments of the invention.

The y axis of the line 804 represents the yield of the protein from thecell homogenate ruptured and obtained from the rupturing device 460, 560or 660 prepared under different pH conditions (X-axis) from anaerobicbacterial cells within the cell-containing suspension with a density of10 g/L, according to one or more embodiments of the invention.

The y axis of the line 806 represents the yield of the protein from thecell homogenate ruptured and obtained from the rupturing device 460, 560or 660 prepared under different pH conditions (X-axis) from anaerobicbacterial cells within the cell-containing suspension with a density of5 g/L plotted against different pH conditions, according to one or moreembodiments of the invention.

The y axis of the line 808 represents the yield of proteins from thecell homogenate ruptured and obtained from the rupturing device 460, 560or 660 a prepared under different pH conditions (X-axis) from anaerobicbacterial cells within the cell-containing suspension with a density of1 g/L, according to one or more embodiments of the invention.

The cell-containing suspension selected for undergoing the rupturingprocess inside the rupturing device 460, 560 or 660 can be of differentpH value, within a range of 1 to 14. The pH value of the cell-containingsuspension can be adjusted by various methods, including acid, base, orsalt addition, or combinations thereof. Here in FIG. 8A, the pH value ofcell-containing suspension is adjusted to approximately 6 or 8 using0.5M sodium hydroxide. The pH value of the cell-containing suspensioncan be adjusted before cell-containing suspension being ruptured insidethe rupturing device 460, 560 or 660. For example, the pH value of thecell-containing suspension can be adjusted inside the cell-containingholding tank 445, 545, or 645.

Example 18. Effect of pH on High Concentrations on Protein-ContainingSuspensions

Samples were collected from a high cell density fermentation vessel andconcentrated to 3-fold by centrifuging cells for 10 minutes at 4,000RPM. Cell pellets were re-suspended in culture media. The culture mediawas adjusted to pH between 5 and 10 using concentrated sodium hydroxide.For example, samples were concentrated to 45 g/L by centrifuging cellsfor 10 minutes at 4,000 RPM. Cell pellets were resuspended in culturemedia. The resulting cell mixture pH was modified to 5, 6, 7, or 8 using0.5M sodium hydroxide. Samples were processed at 15,000 PSI through themicrofluidizer for one pass. Each sample was spun down at 13,300 RPM for6 minutes. The supernatant was collected and a Lowry-based protein assaywas used to determine protein concentration in the soluble fraction.

FIG. 8B illustrates a chart of the yield of soluble protein obtainedfrom the rupturing device 460, 560 or 660 and from cell membranes of theanaerobic bacterial cells within the cell-containing suspension,according to one or more embodiments of the invention.

In FIG. 8B, the rupturing devices selected to conduct the rupturingprocess is a microfluidizer. In FIG. 8B, the rupturing process isconducted to the pressure between 10,000 and 20,000 psi against thecell-containing suspension for one or multiple passes. The supernatantwas collected via centrifugation at 13,300 RPM and the protein wasanalyzed using a Lowry-based protein assay. Table 14 indicates thatincreasing the pH of the culture broth from 4-5 to 6-10 significantlyincreases the percent of protein that is recovered in the solublefraction.

TABLE 14 Recovery of Ruptured Proteins Purified form from 45 g/L ofcells (protein concentration as indicated in mg/mL) Proteinconcentration Sample pH of cultured broth (mg/mL) 1 4-5 7.7 2 6-7 16 37.1-8   18.1 4 8.1-10  18.4

The y axis of the line 812 represents the effect of pH on the yield ofprotein from the cell homogenate ruptured and obtained from therupturing device 460, 560 or 660 prepared under different pH conditions(X-axis) from anaerobic bacterial cells within the cell-containingsuspension with a density of 45 g/L from a change in pH value of thecell-containing suspension, according to one or more embodiments of theinvention.

While the foregoing is directed to embodiments of the present invention,other and further embodiments of the invention may be devised withoutdeparting from the basic scope thereof, and the scope thereof isdetermined by the claims that follow.

What is claimed:
 1. A composition of a protein-rich nutrient supplementgenerated from a fermentation process using anaerobic bacteria,comprising: a protein-containing portion fractionated from a homogenate,wherein the protein-containing portion includes a protein content ofbetween about 50% to about 95% of the composition, a carbohydratecontent of between about 5% to about 35% of the composition, and anucleic acid content of less than about 15% of the composition, whereinthe homogenate comprises the protein-containing portion and aprotein-containing cell debris portion, and is obtained from rupturing acell-containing suspension containing cells of the anaerobic bacteria,wherein the pH of the cell-containing suspension containing cells of theanaerobic bacteria is adjusted to 7.5 or more before the rupturing,wherein the anaerobic bacteria is selected from the group consisting ofClostridium autoethanogenum (DSM 10061 of DSMZ Germany), Clostridiumcoskatii (ATCC PTA-10522), Clostridium ljungdahlii PETC (ATCC 49587),Clostridium liungdahlii ERI2 (ATCC 55380), Clostridium ljungdalilii C-01(ATCC 55988), Clostridium ljungdahlii O-52 (ATCC 55889), Acetobacteriuinwoodii and mixtures thereof, wherein the cell-containing suspensioncontaining the cells of the anaerobic bacteria is obtained fromseparating a fermentation liquid broth being delivered out of afermentation vessel during fermentation of a gaseous substrate by theanaerobic bacteria, and wherein the fermentation liquid broth isseparated into the cell-containing suspension containing the cells ofthe anaerobic bacteria and a cell-free permeate solution, and thecell-containing suspension containing the cells of the anaerobicbacteria has a cell concentration of 18.5 g/L or more.
 2. Thecomposition of claim 1, wherein the gaseous substrate includes one ormore gases comprising carbon monoxide (CO), hydrogen gas (H2), andcarbon dioxide (CO2).
 3. The composition of claim 1, wherein the celldebris portion comprises cell wall particulates, cell membraneparticulates, protein aggregates, inclusion bodies, nucleic acid, andother components of an anaerobic bacterial cell.
 4. The composition ofclaim 1, wherein the fermentation liquid broth delivered out of thefermentation vessel is separated into a cell-free permeate solution andthe cell-containing suspension, wherein the cell-free permeate solutioncomprises a hydrocarbon compound selected from the group consisting ofethanol, butanol, acetic acid, butyric acid, and combinations thereof.5. The composition of claim 1, wherein the nucleic acid content of theprotein-containing portion is less than 2% of the composition.
 6. Thecomposition of claim 1, wherein a protein content in theprotein-containing portion is greater than a carbohydrate content in theprotein-containing, portion.
 7. A composition of a protein-rich nutrientsupplement generated from a fermentation process using an anaerobicbacteria, comprising: a protein-containing portion fractionated from ahomogenate, wherein the protein-containing portion includes a proteincontent of between about 50% to about 95% of the composition, acarbohydrate content of between about 5% to about 35% of thecomposition, and a nucleic acid content of less than about 15% of thecomposition, wherein the homogenate comprises the protein-containingportion and a protein-containing cell debris portion, and is obtainedfrom rupturing a cell-containing suspension containing cells of theanaerobic bacteria, wherein the pH of the cell-containing suspensioncontaining cells of the anaerobic bacteria is adjusted to 7.5 or morebefore the rupturing, wherein the anaerobic bacteria is selected fromthe group consisting of Clostridium autoethanogenum (DSM 10061 of DSMZGermany), Clostridium coskatii (ATCC PTA-10522), Clostridium ljungdahliiPETC (ATCC 49587), Clostridium ljungdahlii ERI2 (ATCC 55380),Clostridium ljungdahlii C-01 (ATCC 55988), Clostridium ljungdahlii O-52(ATCC 55889), Acetobacterium woodii and mixtures thereof, wherein thecell-containing suspension containing the cells of the anaerobicbacteria is obtained from separating a fermentation liquid broth beingdelivered out of a fermentation vessel during fermentation of a gaseoussubstrate by the anaerobic bacteria, and the cell-containing suspensioncontaining the cells of the anaerobic bacteria has a cell concentrationof 18.5 g/L, or more.
 8. A composition of a protein-rich nutrientsupplement generated from a fermentation process using an anaerobicbacteria, comprising: a protein-containing portion fractionated from ahomogenate, wherein the protein-containing portion includes a proteincontent of between about 50% to about 95% of the composition, acarbohydrate content of between about 5% to about 35% of thecomposition, and a nucleic acid content of less than about 15% of thecomposition, wherein the homogenate is obtained from rupturing cellmembranes of the anaerobic bacteria within a cell-containing suspensioncontaining cells of the anaerobic bacteria, wherein the pH of thecell-containing suspension containing cells of the anaerobic bacteria isadjusted to 7.5 or more before the rupturing, wherein the anaerobicbacteria is selected from the group consisting of Clostridiumautoethanogenum (DSM 10061 of DSMZ Germany), Clostridium coskatii (ATCCPTA-10522), Clostridium ljungdahlii PETC (ATCC 49587), Clostridiumljungdahlii ERI2 (ATCC 55380), Clostridium ljungdahlii C-01 (ATCC55988), Clostridium ljungdahlii O-52 (ATCC 55889), Acetobacterium woodiiand mixtures thereof, and wherein the cell-containing suspension isobtained from a fermentation liquid broth being delivered out of afermentation vessel during fermentation of a gaseous substrate by theanaerobic bacteria and separating the fermentation liquid broth into acell-free permeate solution and the cell-containing suspension, and thecell-containing suspension containing the cells of the anaerobicbacteria has a cell concentration of 18.5 g/L or more.
 9. Thecomposition of claim 8, wherein the gaseous substrate includes one ormore gases selected from the group consisting of carbon sourcesubstrates, carbon monoxide (CO), carbon dioxide (CO₂), hydrogen gas(H₂), syngas, and combinations thereof.
 10. The composition of claim 8,wherein the homogenate further comprises a protein-containing celldebris portion, and the protein-containing cell debris portion comprisescell wall particulates, cell membrane particulates, protein aggregates,inclusion bodies, nucleic acid, and other components of an anaerobicbacterial cell.
 11. The composition of claim 8, wherein theprotein-containing portion comprises a nucleic acid content that is lessthan about 12% of the composition.
 12. The composition of claim 8,wherein the separating of the fermentation liquid broth into thecell-free permeate solution and the cell-containing suspension isperformed by ultrafiltration.
 13. The composition of claim 8, whereinthe rupturing of the cell membranes of the anaerobic bacteria within thecell-containing suspension is accomplished by one or more rupturingdevices selected from the group consisting of a microfluidics device, asonication device, an ultrasonic device, a mechanical disruption device,a French press, a freezer, a heater, a pasteurization device, an UVsterilization device, a gamma ray sterilization device, a reactor, ahomogenizer, and combinations thereof.
 14. The composition of claim 8,wherein the protein-containing portion is obtained from separating thehomogenate using a fractionator selected from the group consisting of asolid-liquid fractionator, a centrifugation device, a continuouscentrifuge, a decanter centrifuge, a disc-stack centrifuge, a filtrationdevice, a hollow fiber filtration system, a spiral wound filtrationsystem, a ceramic filter system, a cross-flow filtration device, a sizeexclusion device, one or series of size exclusion columns, one or seriesion exchange columns, one or series of carbon polymer columns, aflow-through magnetic fractionator, and combinations thereof.